Dairy Chemistry and Biochemistry

10.2 Indigenous Enzymes of Bovine Milk 385 gel permeation chromatography, formed the basis of all early methods for the isola- tion of AlP from milk; chromatography of n-butanol extracts of MFGM on Concanavalin-A Agarose/Sepharose 4B/Sephacryl S-200 has been used in recent methods.AlP is optimally active at pH 10.5 when assayed on p-­nitrophenylphosphate but at ~6.8 on caseinate; its optimum temperature is ~37 °C. It is a homodimer of 85 kDa monomers; it contains four atoms of Zn which are essential for activity and is also activated by Mg2+, AlP is inhibited by metal chelators; the apo-enzyme may be reactivated by Zn and a number of other metals, which is used as the principle of methods for determination of very low concentrations of zinc in biological systems. It is also inhibited by inorganic phosphate. The amino acid composition of milk AlP is known but its amino acid sequence has not been reported. The indigenous AlP in milk is similar to the AlP in mammary tissue. The AlP in human milk is similar, but not identical, to human liver AlP (i.e., tissue non-specific type). It has been suggest that there are two AlPs in milk, one of which is from sloughed-off mycoepithelial cells, the other originating from lipid microdroplets and acquired intra-cellularly, which is probably the AlP found in the MFGM. Most or all studies on milk AlP have been on that isolated from cream/MFGM, i.e., the minor form of AlP in milk. Assay Methods In 1935, H.D. Kay and W.R. Graham developed a method based on the inactivation of AlP for determination of the adequate pasteurisation of milk. The principle of this method is still used throughout the world and several modifications have been published. The usual substrates are phenylphosphate, p-nitrophenyl phosphate, phe- nolphthalein phosphate or fluorophos, which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol, phenolphthalein or Fluoroyellow, respectively: XO O H2O H2PO4- + XOH P OH O- where XOH is phenol, p-nitrophenol, phenolphthalein or Fluoroyellow. The liber- ated phosphate could be measured but the increase is small against a high back- ground of inorganic phosphate in milk; therefore, the liberated alcohol is quantified. Reflecting the widespread assay of AlP in routine dairy laboratories, many assay methods have been developed. Reactivation of Alkaline Phosphatase Much work has focussed on ‘phosphatase reactivation’, first recognised in 1953 by R.C. Wright and J. Tramer, who observed that UHT-treated milk was phosphatase-­ negative immediately after processing but became positive on storage; microbial phosphatase was shown not to be responsible. HTST-pasteurised bulk milk does not undergo reactivation, although some samples from individual cows may.

386 10  Enzymology of Milk and Milk Products HTST pasteurisation after UHT treatment usually prevents reactivation, which is never observed in in-container sterilized milk. Reactivation can occur following heating at a temperature as low as 84 °C for milk or 74 °C for cream. The optimum storage temperature for reactivation is 30 °C, at which reactivation is detectable after 6 h and may continue for up to 7 days. The greater reactivation in cream than in milk may be due to protection of the enzyme by fat. A number of attempts have been made to explain the mechanism of reactivation of AlP. The enzyme which becomes reactivated is membrane-bound and several fac- tors which influence reactivation have been established. Mg2+ and Zn2+ strongly pro- mote reactivation but Sn2+, Cu2+, Co2+ and EDTA are inhibitory, while Fe2+ has no effect. Sulphydryl (SH) groups are essential for reactivation; perhaps this is why phosphatase becomes reactivated in UHT milk but not in HTST milk. The role of -SH groups, supplied by denatured whey proteins, is considered to be chelation of heavy metals, which would otherwise react with SH groups of the enzyme (also activated on denaturation), thus preventing renaturation. It has been proposed that Mg2+ or Zn2+ cause a conformational change in the denatured enzyme, which is nec- essary for renaturation. Maximum reactivation occurs in products heated at ~104 °C, adjusted to pH 6.5, containing 64 mM Mg2+ and incubated at 30 °C; homogenisation of products before heat treatment reduces the extent of reactivation. Reactivation of alkaline phosphatase is of considerable practical significance since regulations for HTST pasteurisation specify the absence of phosphatase activ- ity. Methods for distinguishing between renatured and residual native alkaline phos- phatase are based on the increase in phosphatase activity resulting from addition of Mg2+ to the reaction mixture. Significance of AlP Alkaline phosphatase in milk is significant mainly because it is used universally as an index of HTST pasteurisation. However, the enzyme may not be the most appro- priate for this purpose because: • Reactivation of alkaline phosphatase under certain conditions complicates inter- pretation of the test. • The enzyme appears to be fully inactivated by temperature × time combinations (e.g., 70 °C × 16 s) less severe than full HTST conditions. • The relationship between log10 % initial activity and pasteurisation equivalent is less linear than the relationship for lactoperoxidase or γ-glutamyl transpeptidase. Although AlP can dephosphorylate casein under suitable conditions, as far as is known, it has no direct technological significance in milk. Perhaps its pH optimum is too far removed from that of milk, especially in fermented milk products, although the pH optimum on casein is reported to be ~7. Moreover, it is inhibited by inorganic phosphate. Proteolysis is a major contributor to the flavour and texture of cheese. Most of the small water-soluble peptides in cheese are derived from the N-terminal half of αs1- or β-casein; many are phosphorylated and show evidence of phosphatase activity

10.2 Indigenous Enzymes of Bovine Milk 387 (i.e., they are partially dephosphorylated) possibly by indigenous acid phosphatase and bacterial phosphatases are probably responsible. Further work on the signifi- cance of indigenous alkaline and acid phosphatases in the dephosphorylation of phosphopeptides in cheese is warranted. 10.2.4.2  A cid Phosphatase (EC 3.1.3.2) The occurrence of an acid phosphomonoesterase (AcP) in milk was first reported by C. Huggins and P. Talaly in 1948 and confirmed by J. E. C. Mullen, who reported that AcP is optimally active at pH 4.0 and is very heat-stable (heating at 88 °C for 10 min is required for complete inactivation). The enzyme is not activated by Mg2+ (as is AlP), but it is activated slightly by Mn2+ and is strongly inhibited by fluoride. The level of AcP activity in milk is only ~2 % that of AlP; activity reaches a maxi- mum 5–6 days post partum. Isolation and Characterization About 80 % of the AcP in milk is in the skimmed milk but the specific activity is higher in cream; it is strongly attached to the MFGM and is not released by non-­ionic detergents. Acid phosphatase in milk has been purified to homogeneity by various forms of chromatography. Adsorption onto Amberlite IRC50 resin is a very effective first step in purification but only ~50 % of the total AcP in skim milk is adsorbed by Amberlite IRC50, even after re-extracting the skim milk with fresh batches of Amberlite, suggesting that skim milk contains at least two AcP isozymes. About 40 % of the AcP in skim milk partitions into the whey on rennet coagulation; this enzyme, which does not adsorb on Amberlite IRC50, has been partly purified. The AcP isolated from skim milk by adsorption on Amberlite IRC50 has been well char- acterised. It is a glycoprotein with a molecular weight of ~42 kDa and a pI of 7.9. It is inhibited by many heavy metals, F−, oxidising agents, orthophosphates and poly- phosphates and activated by thiol-reducing agents and ascorbic acid; it is not affected by metal chelators. It contains a high level of basic amino acids and no methionine Since milk AcP is quite active on phosphoproteins, including caseins, it has been suggested that it is a phosphoprotein phosphatase. Although casein is a substrate for milk AcP, the major caseins, in the order αs (αs1 + αs2) > β > κ, are competitive inhibi- tors when assayed on p-nitrophenylphosphate, probably due to binding of the enzyme to the casein phosphate groups (the effectiveness of the caseins as inhibitors is related to their phosphate content). Assay Methods Acid phosphatase may be assayed at pH ca. 5, on the same substrates as used for AlP. If p-nitrophenol phosphate or phenolphthalein phosphate is used, the pH must be adjusted to >8 after incubation to induce the colour of p-nitrophenol or phenolphthalein.

388 10  Enzymology of Milk and Milk Products Significance of AcP Although AcP is present in milk at a much lower level than AlP, its greater heat stability and lower pH optimum may make it technologically significant. Dephosphorylation of casein markedly changes its physico-chemical properties. As discussed under AlP, several small partially-dephosphorylated peptides have been isolated from cheese and AcP may be involved. Dephosphorylation may be rate-­ limiting for proteolysis in ripening cheese since most proteinases and peptidases are inactive on phosphoproteins or phosphopeptides. The suitability of AcP as an indicator enzyme for super-pasteurization of milk has been assessed but it is not as suitable for this purpose as γ-glutamyl transferase or lactoperoxidase. 10.2.5  Ribonuclease RNase occurs in various tissues and secretions, including milk. Bovine pancreatic RNase A has been studied in detail; it was the first enzyme to have its complete amino acid sequence determined. It contains 124 amino acid residues, with a calcu- lated MW of 13,683 Da, and has a pH optimum of 7.0–7.5. The first study on the indigenous RNase in milk is that of E.W. Bingham and C. A. Zittle in 1962, who reported that bovine milk contains a much higher level of RNase than the blood serum or urine of human, rat or guinea pig, and that most or all of the activity is in the serum phase; bovine milk could potentially serve as a commercial source of RNase. Like pancreatic RNase, the RNase in milk is opti- mally active at pH 7.5 and is more heat-stable at acid pH values than at pH 7; in acid whey, adjusted to pH 7, 50 % of RNase activity is lost on heating at 90 °C for 5 min and 100 % after 20 min, but it is completely stable in whey at pH 3.5 when heated at 90 °C for 20 min. The enzyme was purified 300-fold by adsorption on Amberlite IRC-50 resin and desorption by 1 M NaCl, followed by precipitation with cold (4 °C) acetone (46–66 % fraction). Elution from Amberlite IRC-50 using a NaCl gradient resolves two isoenzymes, A and B, of RNase in milk, at a ratio of about 4:1, as for pancreatic RNase. Amino acid analysis, electrophoresis and immunological studies showed that milk RNase is identical to pancreatic RNase. It is presumed that the RNase in milk originates in the pancreas and is absorbed through the intestinal wall into the blood, from which it enters milk; however, the level of RNase activity in milk is considerably higher than in blood serum, suggesting active transport. Bovine and buffalo milks contain about equal amounts of RNase and about three times as much as human, ovine or caprine milk and porcine milk contains a very low level of RNase. A high molecular weight (80 kDa) RNase (hmRNase) has been purified from human milk and characterised as a single-chain glycoprotein, with a pH optimum in the range 7.5–8.0. It has been suggested that hmRNase is synthe- sised in the mammary gland and passes into milk, rather than being transferred from blood, as are RNase A and B.

10.2 Indigenous Enzymes of Bovine Milk 389 Little or no RNase activity survives UHT sterilisation (121 °C for 10 s) but about 60 % survives heating at 72 °C for 2 min or at 80 °C for 15 s. RNase activity in raw or heat-treated milk is stable to repeated freezing and thawing and to frozen storage for at least a year. Although RNase has no technological significance in milk, which contains very little RNA, it may have significant biological functions. 10.2.6  L ysozyme (EC 3.1.2.17) The presence of natural antibacterial factor(s) in fresh raw bovine milk was reported by Kitasoto in 1889 and by Fokker in 1890; these inhibitors are now called lactenins, one of which is lactoperoxidase. In 1922, A. Fleming identified an antibacterial agent, shown to be an enzyme, in nasal mucus, tears, sputum, saliva and other body fluids which caused lysis of many types of bacteria (Micrococcus lysodeikiticus was used for assays), which he called lysozyme. Chicken egg white is the richest source of lyso- zyme which constitutes ~3.5 % of egg white protein which is the principal commercial source of lysozyme. Milk was not among the several fluids in which Fleming found lysozyme but shortly afterwards the milk of several species was shown to contain lysozyme; human milk is a comparatively rich source but bovine milk is a poor source. Lysozyme (muramidase, mucopeptide N-acetyl-muramyl hydrolase) hydrolyses the β(1 → 4)-linkage between muramic acid and N-acetylglucosamine of mucopoly- saccharides in the cell wall of certain bacteria, resulting in cell lysis. Egg white lysozyme (EWL) has been studied extensively. Lysozyme was isolated from human milk in 1961 by P. Jolles and J. Jolles, who believed that bovine milk was devoid of lysozyme. Lysozyme was soon found in the milk of several species; the milk of cattle, goats, sheep, pigs and guinea pigs contain a low and variable level of lysozyme. Human and equine milk contain ~400 mg L−1 and ~800 mg L−1 lysozyme, respectively (3,000 and 6,000 times the level in bovine milk); these levels represent ~4 and ~3 % of the total protein in human and equine milk, respectively Although lysozyme is a lysosomal enzyme, it is found in soluble form in many body fluids (milk, tears, mucus, egg white); the lysozyme in milk is usually isolated from whey. In addition to the lysozyme in human, equine and bovine milk, lysozyme has been isolated and partially characterised from the milk of baboon, camel, buffalo and dog. The properties of these lysozymes are generally similar to those of HML, but there are substantial differences, even between the lysozymes of closely-related species, e.g., cow and buffalo. Lysozyme has been isolated from the milk of a wider range of species than any other milk enzyme, which may reflect the perceived importance of lysozymes as a protective agent in milk or perhaps because it can be isolated from milk relatively easily. The pH optimum of HML, bovine milk lysozyme (BML) and EWL is 7.9, 6.35 and 6.2, respectively. The MW these lysozymes is ~15 kDa. The amino acid sequence of HML and EWL are highly homologous, but there are several differ- ences; HML consists of 130 amino acid residues, compared with 129 in EWL, the extra residue in the former being Val100. The amino acid sequence of equine milk

390 10  Enzymology of Milk and Milk Products lysozyme also consists of 129 amino acid residues, but shows only 51 % homology with HML and 50 % homology with EWL. The amino acid sequence of lysozyme is highly homologous with that of α-lactalbumin (α-la), a whey protein which is an enzyme modifier in the biosynthe- sis of lactose. The gene sequence and three-D structure of α-la and c-type ­lysozymes are similar. α-La binds a Ca2+ in an Asp-rich loop but most c-type lysozymes do not bind a Ca2+, those of equine and canine milk being exceptions. All lysozymes are relatively stable to heat at acid pH values (3–4) but are relatively labile at pH > 7. More than 75 % of the lysozyme activity in bovine milk survives heating at 75 °C × 15 min or 80 °C × 15 s and is affected little by HTST pasteurization Presumably, the physiological role of lysozyme is to act as a bactericidal agent. In the case of milk, it may simply be a ‘spill-over’ enzyme or it may have a definite protective role. If the latter is true, then the exceptionally high level of lysozyme in human, equine and assinine milk may be significant. Breast-fed babies generally suffer less enteric problems than bottle-fed babies. While there are many major compositional and physico-chemical differences between bovine and human milk that may be responsible for the observed nutritional characteristics, the disparity in lysozyme content may be significant. Fortification of bovine milk-based infant for- mulae with EWL, especially for premature babies, has been recommended but feed- ing studies are equivocal on the benefits of this practice; it appears that EWL is inactivated in the human GIT. 10.2.7  N -Acetyl-β-d-Glucosaminidase (EC 3.2.1.30) N-Acetyl-β-d-glucosaminidase (NAGase) hydrolyses terminal, non-reducing N-acetyl-β-d-glucosamine residues from glycoproteins. It is a lysosomal enzyme and originates mainly from somatic cells and mammary gland epithelial cells. Consequently, NAGase activity increases markedly and correlates highly with the intensity of mastitis. A field test for mastitis based on NAGase activity has been developed, using chromogenic N-acetyl-β-d-glucosamine-p-nitrophenol as sub- strate; hydrolysis yields yellow p-nitrophenol. NAGase is optimally active at 50 °C and pH 4.2 and is inactivated by HTST pasteurization. 10.2.8  γ -Glutamyl Transpeptidase (Transferase) (EC 2.3.2.2) γ-Glutamyl transferase (GGT) catalyses the transfer of γ-glutamyl residues from γ-glutamyl-containing peptides: g ­ glutamyl­peptide + X ® peptide + g ­ glutamyl­ X, where X is an amino acid. GGT, which has been isolated from the fat globule membrane, has a molecular mass of about 80 kDa and consists of two subunits of 57 and 26 kDa. It is optimally active at pH 8–9, has a pI of 3.85 and is inhibited by iodoacetate, diisopropylfluoro- phosphate and metal ions, e.g., Cu2+ and Fe3+.

10.2 Indigenous Enzymes of Bovine Milk 391 It plays a role in amino acid transport in the mammary gland. γ-Glutamyl ­peptides have been isolated from cheese but since γ-glutamyl bonds do not occur in milk proteins, their synthesis may be catalysed by GGT. The enzyme is relatively heat stable and has been proposed as a marker enzyme for milks pasteurized in the range 72–80 °C × 15 s. GGT is absorbed from the gastrointestinal tract, resulting in high levels of GGT activity in the blood serum of newborn animals fed colostrum or early breast milk. 10.2.9  Amylases (EC 3.2.1.-) Amylase (diastase) was identified in milk in 1883. During the next 40 years, several workers reported that the milk of several species contains an amylase. The principal amylase in milk is α-amylase (EC 3.2.1.1), with a lesser amount of β-amylase (EC 3.2.1.2); the enzymes partition mainly into skimmed milk and whey. A highly-­ concentrated preparation of α-amylase was obtained from whey by E.J. Guy and R. Jenness in 1958 but there appears to have been no further work on the isolation of amylase from bovine milk. Amylase is quite heat-labile and loss of amylase activity was proposed in the 1930s as an index of the intensity of heat treatment applied to milk. Human milk and colostrum contain 25–40 times more α-amylase than bovine milk; α-amylase has been purified from human milk by gel permeation chromatog- raphy and its stability to pH and pepsin determined. The level of α-amylase in human milk is 15–140 times higher than in blood plasma, suggesting that it is not transferred from blood but is synthesised in the mammary gland. Milk α-amylase is similar to salivary amylase. Since milk contains no starch, the function of amylase in milk is unclear. Human milk contains up to 130 oligosaccharides, at a total concentration up to 15 mg mL−1 but α-amylase can not hydrolyse the oligosaccharides in milk. Since human babies secrete low levels of salivary and pancreatic amylases (0.2–0.5 % of the adult level), the high level of amylase activity in human milk may enable them to digest starch. By hydrolysing the polysaccharides in the cell wall of bacteria, it has been sug- gested that milk amylase may have anti-bacterial activity. The amylase activity of human milk is an active area of research at present but there appears to be little or no recent research on the amylase in bovine milk or that of other species. 10.2.10  C atalase (EC 1.11.1.6) Catalase (H2O2:H2O2 oxidoreductase; EC 1.11.1.6) catalyses the decomposition of H2O2, as follows: 2H2O2 ® 2H2O + O2 Catalase activity may be determined by quantifying the evolution of O2 mano- metrically or by titrimetrically measuring the reduction of H2O2. Catalases are haem-containing enzymes that are distributed widely in plant, microbial and animal tissues and secretions; liver, erythrocytes and kidney are particularly rich sources.

392 10  Enzymology of Milk and Milk Products A catalase was among the first enzymes demonstrated in milk by Babcock and Russell in 1897, who reported that an extract of separator slime (somatic cells and other debris) could decompose H2O2, The catalase activity in milk varies with feed, stage of lactation and especially during mastitis when the level of activity increases markedly, making catalase a useful indicator of mastitis but it is now rarely used for this purpose, determination of somatic cell count, N-acetylglucosaminidase activity or electrical conductivity being usually used. Most, ~70 %, of the catalase in milk is in the skimmed milk but the specific activ- ity in the cream is 12-fold higher than in skimmed milk and the MFGM is usually used as the starting material for the isolation of catalase from milk. Although the level of catalase in milk is relatively high and the enzyme is easily assayed, catalase was not isolated from milk until 1983, when O. Ito and R. Akuzawa purified cata- lase from milk and crystallised the enzyme, which had a MW of 225 kDa (by gel permeation). Bovine liver catalase is a homotetramer of 60–65 kDa subunits (total MW ~250 kDa); it seems likely that the structure of catalase in milk is similar. Milk catalase is a heme protein with a MW of 200 kDa, and an isoelectric pH of 5.5; it is stable between pH 5 and 10 but rapidly loses activity outside this range. Catalase is relatively heat-labile; heating at 70 °C for 1 h causes complete inactivation. Like other catalases, it is strongly inhibited by Hg2+, Fe2+, Cu2+, Sn2+, CN− and NO3−. It may act as a lipid pro-oxidant via its heme iron. Catalase was among the first indicators of pasteurisation investigated and recently, it has been considered as an indicator of cheese made from sub-pasteurized milk. Although the inactivation of catalase is a useful index of thermisation of milk (it was almost completely inactivated by heating at 65 °C for 16 s), it is not suitable as an index of cheese made from thermised milk owing to the production of catalase in the cheese during ripening, especially by coryneform bacteria and yeasts, if present. 10.2.11  Lactoperoxidase (EC 1.11.1.7) Peroxidases, which are widely distributed in plant, animal and microbial tissues and secretions, catalyse the following reaction: 2HA + H2O2 ® A + 2H2O where HA is an oxidisable substrate or a hydrogen donor. Lactoperoxidase (LPO) was first demonstrated in milk by C. Arnold in 1881, using “guajaktinctur” as reducing agent; he reported that the activity of LPO is lost on heat- ing milk at 80 °C. Legislation was introduced in Denmark in 1898 requiring that all skim milk returned by creameries to farmers should be flash (i.e., no holding period) pasteurized at 80 °C. Various tests were proposed to ensure that the milk was ade- quately pasteurised, but the most widely used method was that developed in 1898 by V. Storch, who assayed LPO activity using p-phenylenediamine as reducing agent; this test is still used to identify super-pasteurised milk, i.e., milk heated ≥76 °C for 15 s. Work on the isolation of LPO was commenced by S. Thurlow in 1925 and LPO was isolated and crystallised by H. Theorell and A. Aokeson in 1943. It is a heam

10.2 Indigenous Enzymes of Bovine Milk 393 protein containing protoporphyrin IX with 0.069 % Fe, a Soret band at 412 nm, an A412:A280 ratio of 0.9, has a mass of 82 kDa and occurs as two isozymes, A and B. Since then, several improved methods for the isolation of LPO have been pub- lished and its characteristics refined. Since LPO is cationic at the pH of milk, as are lactoferrin and some minor proteins, it can be easily isolated from milk or whey using a cationic exchange resin (e.g., Amberlite CG-50-NH4). There are ten iso- zymes of LPO, arising from differences in the level of glycosylation and deamination of Gln or Asn. LPO consists of 612 amino acids and shows 55, 54 and 45 % identity with human myeloperoxidase, eosinophil peroxidase and thyroperoxidase, respec- tively. LPO binds a Ca2+, which has a major effect on its stability, including its heat stability; at a pH below ~5.0, the Ca2+ is lost, with a consequent loss of stability. LPO is synthesised in the mammary gland and is the second most abundant enzyme in milk (next to xanthine oxidoreductase), constituting ~0.5 % of the total whey proteins (~0.1 % of total protein; 30 mg L−1). Human milk contains mainly myeloperoxidase, with a low level of LPO. Apparently, human colostrum contains a high level of myeloperoxidase, derived from leucocytes, and a lower level of LPO. The level of myeloperoxidase decreases rapidly post partum and LPO is the principal peroxidase in mature human milk. 10.2.11.1  S ignificance of LPO Apart from its exploitation as an index of flash or super-HTST pasteurization, LPO is technologically significant for a number of other reasons also. i. It is a possible index of mastitic infection but is not well correlated with somatic cell count. ii. LPO causes non-enzymic oxidation of unsaturated lipids, acting through its heme group; the heat-denatured enzyme is more active than the native enzyme. iii. Milk contains bacteriostatic or bactericidal substances referred to as lactenins, one of which is LPO, which requires H2O2 and thiocyanate (SCN−) to cause inhi- bition. The nature, mode of action and specificity of the LPO-SCN−-H2O2 system has been widely studied. LPO and thiocyanate, which is produced in the rumen by enzymic hydrolysis of thioglycosides from Brassica plants, occur naturally in milk, but H2O2 does not occur naturally in milk. However, H2O2 can be generated metabolically by catalase-negative bacteria, produced in situ through the action of exogenous glucose oxidase on glucose, or it may be added directly. In the presence of low levels of H2O2 and SCN−, LPO exhibits very potent bactericidal activity; this system is 50–100 times more effective than H2O2 alone. The LPO system has been found to have good bactericidal efficiency for the cold pasteurization of fluids or sanitization of immobilized enzyme col- umns. A self-contained LPO-H2O2-SCN− system using coupled β-galactosidase and glucose oxidase, immobilized on porous glass beads, to generate H2O2 in situ from lactose in milk containing 0.25 mM thiocyanate has been developed. Indigenous xanthine oxidoreductase, acting on added hypoxanthine, may also be exploited to produce H2O2. The bactericidal effects of the LPO-H2O2-SCN− system may be used to cold pasteurize milk in situations where refrigeration

394 10  Enzymology of Milk and Milk Products and/or thermal pasteurization is lacking. Addition of isolated LPO to milk replacers for calves or piglets reduces the incidence of enteritis. iv. It has been proposed that the LPO system may be exploited for bleaching coloured whey. 10.2.12  Xanthine Oxidoreductase (XOR) [EC, 1.13.22; 1.1.1.204] In 1902, F. Schardinger showed that milk contains an enzyme capable of oxidising aldehydes to acids, accompanied by the reduction of methylene blue; this enzyme was commonly called the “Schardinger enzyme”. In 1922, it was shown that milk contains an enzyme capable of oxidising xanthine and hypoxanthine, with the con- comitant reduction of O2 to H2O2, this enzyme was called xanthine oxidase (XO). In 1938, V. H. Booth showed that the Schardinger enzyme is, in fact, xanthine oxidase and partially purified it. XO requires FAD for catalytic activity. Under certain cir- cumstances XO can dehydrogenate xanthine and is now called xanthine oxidoreduc- tase (XOR). XOR exists as two forms, xanthine oxidase (XO; EC 1.1.3.22) and xanthine dehydrogenase (XDH; 1.1.1.204) which can be inter-converted by sulphy- dryl reagents and XDH can be converted irreversibly to XO by specific proteolysis. XOR is concentrated in the MFGM, in which it is the second most abundant protein, after butyrophilin; it represents ~20 % of the protein of the MFGM (~0.2 % of total milk protein; ~120 mg L−1). Therefore, all isolation methods use cream as the starting material; the cream is washed and churned to yield a crude MFGM preparation; dissociating and reducing agents are used to liberate XOR from mem- brane lipoproteins and some form of chromatography is used for purification. Milk XOR is a homodimer of 146 kDa sub-units, each containing ~1,332 amino acid resi- dues; each monomer contains 1 atom of Mo, 1 molecule of FAD and 2 Fe2S2 redox centres. NADH acts as a reducing agent and the oxidation products are H2O2 and O2-. . Cows deficient in Mo have low XOR activity. The quaternary structure of XDH and XO has been described. Milk is a very good source of XOR, at least part of which is transported to the mammary gland via the blood stream. 10.2.12.1  Activity in Milk and Effect of Processing Various processing treatments affect the XOR activity of milk. Activity is increased by ~100 % on storage at 4 °C for 24 h, by 50–100 % on heating at 70 °C for 5 min and by 60–90 % on homogenization. These treatments cause the transfer to XO from the fat phase to the aqueous phase, rendering the enzyme more active. The heat stability of XOR is very dependent on whether it is a component of the fat globules or is in the aqueous phase;. XOR is most heat stable in cream and least in skim milk. Homogenization of concentrated milk prepared from heated milk (90.5 °C for 15 s) partially reactivates XOR, which persists on drying the concentrate, but no reactiva- tion occurs following more severe heating (93 °C for 15 s).

10.2 Indigenous Enzymes of Bovine Milk 395 The XOR activity in human milk is low because 95–98 % of the enzyme mole- cules lack Mo. The level of XOR activity in goat and sheep milk is also low and be increased by supplementing the diet with Mo. 10.2.12.2  Assay Xanthine oxidase activity can be assayed manometrically, potentiometrically, polarographically or spectrophotometrically. The latter may involve the reduction of colourless triphenyltetratetrazolium chloride to a red product or the conversion of xanthine to uric acid which is quantified by measuring absorbance at 290 nm. 10.2.12.3  S ignificance of Xanthine Oxidase • As an index of heat treatment: XOR has been considered as a suitable indicator of milk heated in the temperature range 80–90 °C but the natural variability in the level of XOR activity in milk is too high for its use as a reliable index of heat treatment. • Lipid oxidation: XOR can excite stable triplet oxygen (3O2) to singlet oxygen (1O2), a potent pro-oxidant. Some individual-cow milk samples, which undergo spontaneous oxidative rancidity, contain about ten times the normal level of XOR, and spontaneous oxidation can be induced in normal milk by the addition of XOR to ~4× the normal level. Heat-denatured or FAD-free enzyme is not a pro-oxidant • Atherosclerosis: It has been suggested that XOR enters the vascular system from homogenised milk and may be involved in atherosclerosis via oxidation of plas- malogens in cell membranes; this aspect of XOR attracted considerable attention in the early 1970s but the hypothesis has been discounted. • Reduction of nitrate in cheese: Sodium nitrate is added to milk for many cheese varieties to prevent the growth of Clostridium tyrobutyricum, which causes ­flavour defects and late gas blowing; XOR reduces nitrate to nitrite, which is bactericidal, and then to NO. • Production of H2O2: The H2O2 produced by the action of XOR can serve as a substrate for lactoperoxidase in its action as a bactericidal agent. • Purine catabolism XOR catalyses the catabolism of purines and may be involved in the regulation of blood pressure. • Bactericidal activity: XOR has strong antibacterial activity in the human intes- tine, probably via the production of peroxynitrite (ONOO−). • Secretion of milk fat: Probably the most important role of XOR in milk is now considered to be in the secretion of milk fat globules from the mammary secre- tory cells. The triglycerides in milk are synthesised in the endoplasmic retricu- lum (ER), which is located toward the basal membrane of the cell. In the ER, the TGs are formed into micro lipid droplets and released through the involvement of the protein, acidophilin (ADPH), which surrounds the globules. The ADPH-­ covered globules move toward the apical membrane of the cell, probably through

396 10  Enzymology of Milk and Milk Products a microtubular/microfilament system, and acquire additional coat material, cyto- plasmic proteins and phospholipids. At the apical membrane, ADPH forms a disulphide-linked complex with butyrophlin (BTN) and dimeric XOR. Somehow, XOR causes blebbing of the fat globule through the membrane and it is eventu- ally pinched off and released into the alveolar lumen. In the secretion of milk fat globules, XOR does not function as an enzyme (see Chap. 3). • The production of reactive oxygen and nitrogen species by XOR and the involve- ment of these in various pathophysiological conditions including cardiovascular disease has attracted much research attention. 10.2.13  Sulphydryl Oxidase (EC 1.8.3.-) Milk contains sulphydryl oxidase (SO), capable of oxidizing sulphydryl groups of cysteine, glutathione and proteins to the corresponding disulphide. The enzyme is an aerobic oxidase which catalyses the following reaction: 2RSH + O2 ® RSSR + H2O2 It undergoes marked self-association and can be purified readily by chromatog- raphy on porous glass. The enzyme has a molecular mass of ~89 kDa, a pH optimum of 6.8–7.0, and a temperature optimum of 35 °C. Its amino acid composi- tion, its requirement for iron but not for molybdenum and FAD, and the catalytic properties of the enzyme, indicate that sulphydryl oxidase is distinct from xanthine oxidoreductase and thiol oxidase (EC 1.8.3.2). SO is capable of oxidizing reduced ribonuclease and restoring enzymatic activ- ity, suggesting that its physiological role may be the non-random formation of pro- tein disulphide bonds during protein biosynthesis. SO immobilized on glass beads has the potential to ameliorate the cooked flavour arising from sulphydryl groups exposed upon protein denaturation. The production of sulphur compounds is believed to be very important in the development of Cheddar cheese flavour. Residual sulphydryl oxidase activity may play a role in reoxidizing sulphydryl groups exposed upon heating cheesemilk; the sulphydryl groups thus protected may be reformed during ripening. 10.2.14  Superoxide Dismutase (EC 1.15.1.1) Superoxide dismutase (SOD) scavenges superoxide radicals, O2-× according to the reaction: 2O2-× + 2H+ ® H2O2 + O2 The H2O2 formed may be reduced by catalase, peroxidase or a suitable reducing agents. SOD occurs in many animal and bacterial cells; its biological function is to protect tissue against oxygen free radicals in anaerobic systems.

10.2 Indigenous Enzymes of Bovine Milk 397 SOD, isolated from bovine erythrocytes, is a blue-green protein due to the pres- ence of copper, removal of which by treatment with EDTA results in loss of activity which is restored by adding Cu2+; it also contains Zn2+, which appears not to be at the active site. The enzyme consists of two identical 16 kDa subunits held together by one or more disulphide bonds. The amino acid sequence has been established. Milk contains trace amounts of SOD which has been isolated and characterized; it appears to be identical to the bovine erythrocyte enzyme. SOD inhibits lipid oxidation in model systems. The level of SOD in milk parallels that of XO (but at a lower level), suggesting that SOD may be excreted in milk in an attempt to offset the pro-oxidant effect of XO. However, the level of SOD in milk is probably insufficient to explain observed differences in the oxidative stability of milk. The possibility of using exog- enous SOD to retard or inhibit lipid oxidation in dairy products has been considered. In milk, SOD is stable at 71 °C for 30 min but loses activity rapidly at even slightly higher temperatures. Slight variations in pasteurization temperature are therefore critical to the survival of SOD in heated milk products and may contribute to variations in the stability of milk to oxidative rancidity. 10.2.15  O ther Enzymes In addition to the enzymes describe above, a number of other indigenous enzymes (Table 10.2) have been isolated and partially characterized. Although fairly high lev- els of some of these enzymes occur in milk, they have no apparent function in milk and will not be discussed further. Many other enzymatic activities have been detected in milk but have not been isolated and limited information on their molecular and biochemical properties in milk is available; some of these are listed in Table 10.3. Table 10.2  Other enzymes that have been isolated from milk and partially characterized but which are of no known significance (see Farkye 2003) Enzyme EC 1.11.1.9 Reaction catalysed Comment Glutathione 2 GSH + H2O ⇌ GSSH Contains Se peroxidase EC 3.1.27.5 Hydrolysis of RNA Ribonuclease Milk a very rich EC 3.2.1.1 Starch source; similar to α-Amylase EC 3.2.1.2 Starch pancreatic RNase β-Amylase EC 3.2.1.24 α-Mannosidase EC 3.2.1.31 Contains Zn2+ β-Glucuronidase EC 3.1.3.5 5′-Nucleotidase 5′ nucleotides + H2O ⇌ Diagnostic test for EC 3.6.1.3 Ribonucleosides + Pi mastitis Adenosine ATP + H2O ⇌ ADP + Pi triphosphatase EC 4.1.2.13 Aldolase Fructose 1,6 diP ⇌ glyceraldehyde-3-P dihydroxyacetone-P

Table 10.3  Partial list of minor enzymes in milk (modified from Farkye 2003) 398 10  Enzymology of Milk and Milk Products Enzyme Reaction catalyzed Source Distribution EC 1.1.1.1 Ethanol + NAD+ ⇌ acetaldehyde + NADH + H+ in milk EC 1.1.1.14 l-Iditol + NAD+ ⇌ l-sorbose + NADH EC 1.1.1.27 Alcohol dehydrogenase Lactic acid + NAD+ ⇌ pyruvate acid + NADH + H+ – SM EC 1.1.1.37 l-Iditol dehydrogenase Malate + NAD+ ⇌ oxaloacetate + NADH EC 1.1.1.40 Lactate dehydrogenase Malate + NADP+ ⇌ pyruvate + CO2 + NADH Mammary gland SM EC 1.1.1.42 Malate dehydrogenase Isocitrate + NADP+ ⇌ 2-oxogluterate + CO2 + NADH Mammary gland SM EC 1.1.1.44 Malic enzyme 6-Phospho-d-­gluconate + NADP+ ⇌ d-ribose-5 Mammary gland SM Isocitrate dehydrogenase phosphate + CO2 + NADPH Mammary gland SM EC 1.1.1.49 Phosphoglucuronate d-Glucose-d-g­ luconate + NADP+ ⇌ d-glucono-1,5-lactone-6- dehydrogenase (decarboxylating) phosphate + NADPH Mammary gland SM EC 1.4.3.6 Glucose-6-phosphate RCH2NH2 + H2O + O2 ⇌ RCHO + NH3 + H2O2 – dehydrogenase Spermine → spermidine → putrescine – SM – Amine oxidase (Cu-containing) Catalyses the transfer of fucose form GDP l-fucose to specific – SM Polyamine oxidase oligosaccharides and glycoproteins – SM EC 1.6.99.3 Fucosyltransferase NADH + acceptor ⇌ NAD+ + reduced acceptor EC 1.8.1.4 Dihydrolipoamide + NAD+ ⇌ lipoamide + NADH – FGM NADH dehydrogenase – SM/FGM EC 2.4.1.22 Dihydrolipoamide dehydrogenase UDP galactose + d-glucose ⇌ UDP + lactose (Diaphorase) Golgi apparatus SM EC 2.4.1.38 Lactose synthetase A protein: UDP galactose + N-acetyl d-glucosaminyl-­glycopeptide  ⇌ UDP-galactose: d-glucose, UDP + 4,β-d-­galactosyl-N-acetyl-d-glucosaminyl glycopeptide – FGM EC 2.4.1.90 1-galactosyltransferase; B protein: UDP galactose + N-acetyl-d-g­ lucosamine ⇌ UDP Golgi apparatus – α-lactalbumin N-acetyllactosamine – SM EC 2.4.99.6 Glycoprotein CMP-N-­acetylneuraminate  +  β-d-­galactosyl 1,4-N-acetyl 4-β-galactosyltransferase d-glucosaminyl glycoprotein ⇌ CMP + α-N-­acetylneuraminyl N-Acetyllactosamine synthase 1-2,3-β-d-galactosyl-1,4-N-­acetyl-d-glucosaminyl-­glycoprotein CMP-N-acetyl-N-acetyl-­ lactosaminide α-2,3-sialyltransferase

EC 2.5.1.3 Thiamine-phosphate 2-Methyl-4-amino-5-h­ ydroxymethyl/pyrimidine – FGM 10.2 Indigenous Enzymes of Bovine Milk pyrophosphorylase diphosphate + 4-methyl-5-(2-­phosphonooxyethyl)- thiazole ⇌ pyrophosphate + thiamine monophosphate Blood SM EC 2.6.1.1 Aspartate aminotransferase l-Aspartate + 2-oxogluterate ⇌ oxaloacetate + l-glutamate Blood SM EC 2.6.1.2 Alanine aminotransferase l-Alanine + 2-oxogluterate ⇌ pyruvate + l-glutamate EC 2.7.5.1 Phosphoglucomutase – SM EC 2.7.7.49 RNA-directed DNA polymerase n Deoxynucleoside triphosphate ⇌ n pyrophosphate + DNAn – SM EC 2.8.1.1 Thiosulphate sulphur transferase Thiosulphate + cyanide ⇌ sulphite + thiocyanate Blood FGM EC 3.1.1.8 Cholinesterase An acylcholine + H2O ⇌ choline + a carboxylic acid anion FGM EC 3.1.3.9 Glucose-6-phosphatase d-Glucose 6-phosphate + H2O ⇌ d-glucose + inorganic phosphate – EC 3.1.4.1 Phosphodiesterase Lysosomes – EC 3.1.6.1 Arylsulphatase Phenol sulphate + H2O ⇌ phenol + sulphate Lysosomes FGM EC 3.2.1.21 β-Glucosidase Hydrolysis of terminal non-reducing β-d-glucose residues FGM EC 3.2.1.23 β-Galactosidase Hydrolysis of terminal non-reducing β-d-galactose residues in Lysosomes β-d-galactosides – – EC 3.2.1.51 α-Fucosidase An α-l-fucoside + H2O ⇌ an alcohol + l-fucose SM EC 3.4.11.1 Cytosol aminopeptidase Aminoacyl-­peptide + H2O ⇌ amino acid + peptide – SM (Leucine aminopeptidase) Cystyl-peptides + H2O ⇌ amino acid + peptide – – SM EC 3.4.11.3 Cystyl-aminopeptidase Hydrolyses peptide bonds, preferentially Lys-X, Arg-X SM/FGM (Oxytocinase) Pyrophosphate + H2O ⇌ 2 orthophosphate – – SM/FGM EC 3.4.21.4 Trypsin A dinucleotide + H2O ⇌ 2 mononucleotides – SM H2CO3 ⇌ CO2 + H2O – SM EC 3.6.1.1 Inorganic pyrophosphatase d-glucose-6-­phosphate  ⇌  fructose-6-­phosphate FGM ATP + acetyl-­CoA + HCO3 ⇌ ADP + orthophosphate + malonyl-CoA EC 3.6.1.1 Pyrophosphate phosphorylase EC 3.6.1.9 Nucleotide pyrophosphate EC 4.2.1.1 Carbonate dehydratase EC 5.3.1.9 Glucose-6-phosphate isomerase EC 6.4.1.2 Acetyl-CoA carboxylase SM skim milk, FGM fat globule membrane 399

400 10  Enzymology of Milk and Milk Products 10.3  E xogenous Enzymes in Dairy Technology 10.3.1  I ntroduction Crude enzyme preparations have been used in food processing since pre-historic times; classical examples are rennets in cheesemaking, malt in brewing and papaya leaves to tenderize meat. Added (exogenous) enzymes are attractive in food pro- cessing because they can induce specific changes in contrast to chemical or physical methods which may cause non-specific undesirable changes. For some applications, there is no alternative to enzymes, e.g., rennet-coagulated cheeses, whereas in some cases, enzymes are preferred over chemical methods because they cause fewer side reactions and consequently give superior products, e.g., hydrolysis of starch. Although relatively few enzymes are used in the dairy industry on a significant scale, the use of rennets in cheesemaking is one of the principal of all industrial applications of enzymes. The applications of exogenous enzymes in dairy technology can be divided into two groups: 1 . Technological, in which an enzyme is used to modify a milk constituent or to improve its microbiological, chemical or physical stability. 2 . Enzymes as analytical reagents. Although the technological applications are quantitatively the more important, many of the analytical applications of enzymes are unique and are becoming increasingly important. Since the principal constituents of milk are proteins, lipids and lactose, protein- ases, lipases and β-galactosidase (lactase) are the principal exogenous enzymes used in dairy technology. Apart from these, there are, at present, only minor applica- tions for glucose oxidase, catalase, superoxide dismutase and lysozyme. Lactoperoxidase, xanthine oxidase and sulphydryl oxidase might also be included, although at present the indigenous form of these enzymes is exploited. 10.3.2  P roteinases There is one major (rennet) and several minor applications of proteinases in dairy technology. 10.3.2.1  R ennets The use of rennets in cheesemaking is the principal application of proteinases in food processing. The sources of rennets and their role in milk coagulation and cheese ripening were discussed in Chap. 12 and will not be considered here.

10.3 Exogenous Enzymes in Dairy Technology 401 10.3.2.2  Accelerated Cheese Ripening Cheese ripening is a slow, expensive and not fully controlled process; consequently, there is increasing interest, at both the research and industrial levels, in accelerating ripening. Various approaches have been investigated to accelerate ripening, includ- ing a higher ripening temperature (especially for Cheddar-type cheese which is usu- ally ripened at 6–8 °C), exogenous proteinases and peptidases, modified starters (e.g., heat-shocked or lactose-negative) and genetically engineered starters or starter adjuncts The possible use of exogenous proteinases and peptidases attracted consid- erable attention for a period but uniform distribution of the enzymes in the cheese curd is a problem, microencapsulation of enzymes offers a possible solution. Exogenous proteinases/peptidases are not used commercially in natural cheeses but are being used to produce “Enzyme Modified Cheese” for use in processed cheese, cheese dips and sauces. Selected genetically modified and adjunct cultures appear to be more promising. 10.3.2.3  Protein Hydrolyzates Protein hydrolyzates are used as flavourings in soups and gravies and in dietetic foods. They are generally prepared from soy, gluten, milk, meat or fish proteins by acid hydrolysis. Neutralization results in a high salt content which is acceptable for certain applications but may be unsuitable for dietetic foods and food supplements. Furthermore, acid hydrolysis causes total or partial destruction of some amino acids. Partial enzymatic hydrolysis is a viable alternative for some applications but bitterness due to hydrophobic peptides is frequently encountered. Bitterness may be eliminated or at least reduced to an acceptable level by treatment with activated carbon, carboxypeptidase, aminopeptidase, ultrafiltration or hydrophobic chroma- tography. Caseins yield very bitter hydrolyzates but the problem may be minimized by the judicious selection of the proteinase(s) and by using exopeptidases (especially aminopeptidases) together with the proteinase. A novel, potentially very significant application of proteinases in milk protein technology is the production of biologically active peptides (see Chap. 11). Carefully selected proteinases of known specificity are required for such applications but the resulting products have high added value. The functional properties of milk proteins may be improved by limited proteoly- sis. Acid-soluble casein, free of off-flavour and suitable for incorporation into bev- erages and other acid foods (in which casein is insoluble) can be produced by limited proteolysis. The antigenicity of casein is destroyed by proteolysis and the hydrolysate is suitable for use in milk protein-based foods for infants allergic to cows’ milk formulations. Controlled proteolysis improves the meltability of directly-acidified cheese but excessive proteolysis causes bitterness. Partial prote- olysis of lactalbumin (heat-coagulated whey proteins), which is insoluble and has

402 10  Enzymology of Milk and Milk Products very poor functional properties, yields a product that is almost completely soluble above pH 6; although the product is slightly bitter, it appears promising as a food ingredient. Limited proteolysis of whey protein concentrate reduces its emulsifying capacity, increases its specific foam volume but reduces foam stability and increases heat stability. 10.3.3  β-Galactosidase β-Galactosidase (lactase; EC 3.1.2.23), which hydrolyses lactose to glucose and galactose, is probably the second most significant enzyme in dairy technology. In the 1970s, β-galactosidase was considered to have very considerable potential but this has not materialized although there are a number of significant technological and nutritional applications. The various aspects of lactose and applications of β-galactosidase are considered in Chap. 2. β-Galactosidase has transferrase as well as hydrolase activity and under certain conditions produces several oligosaccharides which have interesting nutritional and physic-chemical properties. 10.3.4  L ipases The principal application of lipases in dairy technology is in cheese manufacture, particularly certain hard Italian varieties. The characteristic “piccante” flavour of these cheeses is due primarily to short-chain fatty acids resulting from the action of lipase(s) in the rennet paste traditionally used in their manufacture. Rennet paste is prepared from the stomachs of calves, kids or lambs slaughtered after suckling; the stomachs and contents are aged and then macerated. The lipase in rennet paste, pre-­ gastric esterase (PGE), is secreted by a gland at the base of the tongue, which is stimulated by suckling; the secreted lipase is washed into the stomach with the ingested milk. The physiological significance of PGE, which is secreted by several species, is to assist in lipid digestion in the neonate which has limited pancreatic function. PGE has a high specificity for short chain fatty acids, especially butanoic acid, esterified on the sn-3 position of glycerol, although some inter-species differ- ences in specificity have been reported. Semi-purified preparations of PGE from calf, kid and lamb are commercially available and give satisfactory results; slight differences in specificity renders one or other more suitable for particular applica- tions. Connoisseurs of Italian cheese claim that rennet paste gives superior results to semi-purified PGE, and it is cheaper. Rhizomucor miehei secretes a lipase that is reported to give satisfactory results in Italian cheese manufacture. This enzyme has been characterized and is commercially

10.3 Exogenous Enzymes in Dairy Technology 403 available as “Piccantase”. Lipases secreted by selected strains of Penicillium r­ oqueforti and P. candidum are considered to be potentially useful for the manufac- ture of Italian and other cheese varieties. Extensive lipolysis occurs in Blue cheese varieties in which the principal lipase is secreted by P. roqueforti (see Chap. 12). It is claimed that treatment of Blue cheese curd with PGE improves and intensifies its flavour but this practice is not widespread. Several techniques have been developed for the production of fast-­ ripened Blue cheese-type products suitable for use in salad dressings, cheese dips, etc. Lipases, usually of fungal origin, are used in the manufacture of these products or to pre-hydrolyse fats/oils used as ingredients in their production. Although Cheddar cheese undergoes relatively little lipolysis during ripening, it is claimed that addition of PGE, gastric lipase or selected microbial lipases improves the flavour of Cheddar, especially that made from pasteurized milk, and accelerates ripening. It is also claimed that the flavour and texture of Feta and Egyptian Ras cheese can be improved by adding kid or lamb PGE or a low level of selected microbial lipases to the cheese milk, especially if milk concentrated by ultrafiltra- tion is used. Lipases are used to hydrolyze milk fat for a variety of uses in the confectionary, candy, chocolate, sauce and snack food industries and there is interest in using immobilized lipases to modify fat flavours for such applications. Enzymatic inter- esterification of milk lipids to modify rheological properties is also feasible. 10.3.5  L ysozyme As discussed in Sect. 10.2.5, lysozyme has been isolated from the milk of several species; human and equine milks are especially rich sources. In view of its anti-­ bacterial activity, the large difference in the lysozyme content between human and bovine milks may have significance in infant nutrition. It is claimed that supplemen- tation of baby food formulae based on cows’ milk with egg white lysozyme is ben- eficial, especially with premature babies but views on this not unanimous, and it is not used commercially. Nitrate is added to many cheese varieties to prevent the growth of Clostridium tyrobutyricum which causes off-flavours and late gas blowing. However, the use of nitrate in foods is considered to be undesirable because of its involvement in nitro- samine formation and many countries have reduced the permitted level or prohib- ited its use. Lysozyme, which inhibits the growth of vegetative cells of Cl. tyrobutyricum and hinders the germination of its spores, is an alternative to nitrate for the control of late gas blowing in cheese. Lysozyme also kills Listeria spp. Co-immobilized lysozyme has been proposed for self-sanitizing immobilized enzyme columns.

404 10  Enzymology of Milk and Milk Products H HO HHO | | || | | || NC C NC C | | (CH2)2 O=C | O=C | | NH2 NH | + (CH2)4 | NH2 NC C | | | || (CH2)4 HHO | NC C | | || HHO Fig. 10.1  Formation of an ε(γ-glutamyl) lysine isopeptide bond between proteins or peptides 10.3.6  Transglutaminase Transglutaminase (TGase; EC 2.3.1.13; γ-glutamylpeptidase, amine-γ-glutamyl transferase) catalyses the acyl transfer between the γ-carboxyl amine group of a peptide-bound glutamine residue and the primary amine group of various substrates, lysine being of special interest (Fig. 10.1). Because of their open structure the caseins are very good substrates for TGase but native whey proteins are not good. Benefits of using TGase in the dairy industry include: 1. The emulsifying capacity of sodium caseinate is scarcely affected by TGase treatment but the stability of the emulsion is improved, 2. The firmness of fermented milk products is improved by TGase treatment of the milk, 3. The rennet coagulability of milk is adversely affected because the accessibility of κ-casein is hindered 4 . The heat stability of milk is greatly increased and the minimum in the HCT-pH profile is removed. 10.3.7  C atalase (EC 1.1.1.6) Hydrogen peroxide is a very effective chemical sterilant and although it causes some damage to the physico-chemical properties and nutritional value of milk protein, principally by oxidizing methionine, it is used as a milk preservative, especially in

10.3 Exogenous Enzymes in Dairy Technology 405 warm regions lacking refrigeration. Excess H2O2 may be reduced following treatment by soluble exogenous catalase (from beef liver, Aspergillus niger or Micrococcus lysodeiktieus). Immobilized catalase has been investigated for this purpose but the immobilized enzyme is rather unstable. As discussed in Sect. 10.3.8, catalase is frequently used together with glucose oxidase in many of the food applications of the latter; however, the principal poten- tial application of glucose oxidase in dairy technology is for the in situ production of H2O2 for which the presence of catalase is obviously undesirable. 10.3.8  Glucose Oxidase (EC 1.1.3.4) Glucose oxidase (GO) catalyses the oxidation of glucose to gluconic acid (via glu- conic acid-δ-lactone) according to the following reaction: Glucose GO gluconic acid-δ-lactone + FADH2 FAD, O2 H2O O2 GO lactonase FAD + H2O2 or Catalase spontaneous gluconic acid H2O + ½O2 The H2O2 formed is normally reduced by catalase present as a contaminant in commercial GO preparations (from P. notatum, P. glaucum or A. niger) or added separately. Glucose oxidase, which has a pH optimum of ~5.5, is highly specific for d-glucose and may be used to assay specifically for d-glucose in the presence of other sugars. In the food industry, glucose oxidase has four principal applications: 1. Removal of residual trace levels of glucose: This application, which is particu- larly useful for the treatment of egg white prior to dehydration (although alterna- tive procedures using yeast fermentation are used more commonly), is of little, if any, significance in dairy technology. 2 . Removal of trace levels of oxygen: Traces of oxygen in wines and fruit juices cause discolouration and/or oxidation of ascorbic acid. Chemical reducing agents may be used to scavenge oxygen but enzymatic treatment with GO may be preferred. Glucose oxidase has been proposed as an antioxidant system for high-fat products such as mayonnaise, butter and whole milk powder but it does not appear to be widely used for this purpose, probably because of cost vis-à-vis

406 10  Enzymology of Milk and Milk Products chemical antioxidants (if permitted) and the relative effectiveness of inert gas flushing in preventing lipid oxidation in canned milk powder. 3 . Generation of H2O2 in situ: The H2O2 generated by glucose oxidase has a direct bactericidal effect (which appears to be a useful side-effect of GO applied to egg products) but its bactericidal properties can be much more effectively exploited as a component of the lactoperoxidase/H2O2/SCN− system. Glucose required for GO activity may be added or produced by the action of β-galactosidase on lac- tose (both β-galactosidase and glucose oxidase have been immobilized on porous glass beads). H2O2 may also be generated in situ by the action of xanthine o­ xidoreductase on added hypoxanthine. It is likely that exogenous H2O2 will be used in such applications rather than H2O2 generated by glucose oxidase or xanthine oxidase. 4 . Production of acid in situ: Direct acidification of dairy products, particularly Cottage and Mozzarella cheeses, is fairly common. Acidification is normally performed by addition of acid or acidogen (usually gluconic acid-δ-lactone) or by a combination of acid and acidogen. In situ production of gluconic acid from added glucose or from glucose produced in situ from lactose by β-galactosidase or from added sucrose by invertase; immobilized glucose oxidase has been investigated but is not is used commercially for direct acidification of milk. Production of lactobionic acid from lactose by lactose dehydrogenase has also been proposed for the direct acidification of dairy and other foods. 10.3.9  Superoxide Dismutase (EC 1.15.1.1) Superoxide dismutase (SOD), an indigenous enzyme in milk, was discussed in Sect. 10.2.10. A low level of exogenous SOD, coupled with catalase, is a very effec- tive inhibitor of lipid oxidation in dairy products, particularly for preserving the flavour of long-life UHT milk which is prone to lipid oxidation. Obviously, the commercial feasibility of using SOD as an antioxidant depends on cost, particularly vis-à-vis chemical methods, if permitted. 10.3.10  Glucose Isomerase (EC 5.3.1.5) Glucose isomerase, which converts glucose to fructose, is widely use to produce high-fructose syrups from glucose produced from starch by amylase and glucoamy- lase. It has potential in the dairy industry in conjunction with β-galastosidase for the production of galactose-glucose-fructose syrup (which is sweeter than galactose-­ glucose syrup) from lactose.

10.3 Exogenous Enzymes in Dairy Technology 407 10.3.11  E xogenous Enzymes in Food Analysis Exogenous enzymes have several applications in food analysis. One of the principal attractions of enzymes as analytical reagents is their specificity which eliminates the need for extensive clean-up of the sample and makes it possible to quantify separately closely related molecules, e.g., d- and l-glucose, d- and l-lactic acid. Enzymatic assays can be very sensitive; some can detect concentrations at the picomole level. Enzymes can be immobilized as enzyme electrodes and as such can be used continuously to monitor changes in the concentration of a substrate in a product stream. Disadvantages of enzymes as analytical reagents are their relatively high cost, especially when few samples are to be analysed, relatively poor stability (due to denaturation or inhibition) and the need to use highly purified enzymes. Enzymes are rarely used by industrial food laboratories but find regular applica- tion in more specialized analytical or research laboratories. Important applications are summarized in Table 10.4 (information on products and methods may be obtained from R-Biopharm AG at www.r-biopharm.com). There are alternative chemical and/ or physical methods, especially some form of chromatography, for all these applica- tions, but extensive clean-up and perhaps concentration may be required. The use of luciferase to quantify ATP in milk is the principle of modern rapid methods for assessing the bacteriological quality of milk based on the production of ATP by bacteria. Such methods have been automated and mechanized. Table 10.4  Some examples of compounds in milk that can be analysed by enzymatic assays Substrate Enzyme d-Glucose Galactose Glucose oxidase; Glucokinase; Hexokinase Fructose Galactose dehydrogenase Lactose Fructose dehydrogenase Lactulose β-Galactosidase, then analyse for glucose or galactose d- and l-lactic acid β-Galactosidase, then analyse for fucose or galactose Citric acid d- and l-lactic dehydrogenase Acetic acid Citrate dehydrogenase Acetate kinase + pyruvate kinase + lactic Ethanol dehydrogenase Glycerol Alcohol dehydrogenase Fatty acids Glycerol kinase Amino acids Acyl-CoA synthetase + Acyl-CoA oxidase Metal ions (inhibitors or activators) Decarboxylases; Deaminases ATP Choline esterase; Luciferase; Invertase Pesticides (inhibitors) Luciferase Inorganic phosphate Hexokinase; Choline esterase Nitrate Phosphorylase a Nitrate reductase

408 10  Enzymology of Milk and Milk Products 10.3.11.1  Enzyme-Linked Immunosorbent Assays An indirect application of enzymes in analysis is as a marker or label in enzyme-­ linked immunosorbent assays (ELISA). In ELISA, the enzyme does not react with the analyte; instead, an antibody is raised against the analyte (antigen or hapten) and labelled with an easily-assayed enzyme, usually a phosphatase or a peroxidase. The enzyme activity is proportional to the amount of antibody in the system, which in turn is proportional, directly or indirectly depending on the arrangement used, to the amount of antigen present. Either of two approaches may be used: competitive and non-competitive, each of which may be used in either of two modes. 1. Competitive ELISA On the basis of enzyme-labelled antigen The antibody (Ab) is adsorbed to a fixed phase, e.g., the wells of a microtiter plate. An unknown amount of antigen (Ag, analyte) in the sample to be assayed together with a constant amount of enzyme-labelled antigen (Ag-E) are then added to the well (Fig. 10.2). The Ag and Ag-E compete for the fixed amount of Ab and amount of Ag-E bound is inversely proportional to the amount of Ag present in the sample. After washing away the excess of unbound antigen (and other materials), a chromogenic substrate is added and the intensity of the colour determined after incubation for a fixed period. The intensity of the colour is inversely proportional to the concentration of antigen in the sample (Fig. 10.2a). On the basis of enzyme-labelled antibody In this mode, a fixed amount of unlabelled antigen (Ag) is bound to microtiter plates. A food sample containing antigen is added, followed by a fixed amount of enzyme-labelled antibody (Ab-E) (Fig. 10.2b). There is competition between the fixed and free antigen for the limited amount of Ab-E. After an appropriate reac- tion time, unbound Ag (and other materials) are washed from the plate and the amount of bound enzyme activity assayed. As above, the amount of enzyme activ- ity is inversely proportional to the concentration of antigen in the food sample. 2. Non-competitive ELISA The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtiter plates; a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Fig. 10.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed; activity is directly related to the concentration of antigen. Examples of ELISA in dairy analyses include: Quantifying denaturation of β-lactoglobulin in milk products Detection and quantitation of adulteration of milk from one species with that from other species, e.g., sheeps’ milk by bovine milk.

10.3 Exogenous Enzymes in Dairy Technology 409 Fig. 10.2  Schematic representation of a competitive enzyme-linked immunosorbent assay using (a) immobilized antigen or (b) immobilized antibody Authentication of cheese, e.g., sheeps’ milk cheese. Detection and quantitation of bacterial enzymes in milk, e.g., from psychrotrophs. Quantitation of antibiotics. Potential application of ELISA include monitoring proteolysis in the production of protein hydrolyzates or in cheese

410 10  Enzymology of Milk and Milk Products Fig. 10.3  Schematic representation of a non-­competitive enzyme-linked immunosorbent assay using the “sandwich” technique

References and Suggested Reading 411 References and Suggested Reading Indigenous Enzymes in Milk Abd El-Salam, M. H., & El-Shibiny, S. (2011). A comprehensive review on the composition and properties of buffalo milk. Dairy Science and Technology, 91, 663–699. Anderson, M., & Cawston, T. E. (1975). Reviews in the progress of dairy science. The milk fat globule membrane. Journal of Dairy Research, 42, 459–483. Andrews, A. T., Olivecrona, T., Bengtsson-Olivecrona, G., Fox, P. F., Björck, L., & Farkye, N. Y. (1991). Indigenous enzymes in milk. In P. F. Fox (Ed.), Food enzymology (pp. 53–129). London, UK: Elsevier Applied Science. Andrews, A. T., Olivecrona, T., Vilaro, S., Bengtsson-Olivecrona, G., Fox, P. F., Björck, L., et al. (1992). Indigenous enzymes in milk. In P. F. Fox (Ed.), Advanced dairy chemistry (Proteins, Vol. 1, pp. 285–367). London, UK: Elsevier Applied Science. Bastian, E. D., & Brown, R. J. (1996). Plasmin in milk and dairy products, an update. International Dairy Journal, 6, 435–457. Blanc, B. (1982). Les proteines du lait, à activete enzymatique et harmonal. Le Lait, 62, 352–395. Booth, V. H. (1938). The specificity of xanthine oxidase. Biochemical Journal, 32, 494–502. Brockerhoff, H., & Jensen, R. G. (1974). Lipolytic enzymes. New York, NY: Academic. Chandan, R. C., & Shahani, K. M. (1964). Milk lipase: A review. Journal of Dairy Science, 47, 471–480. Corry, A. M. (2004). Purification of bile salts-stimulated lipase from breast milk and ligand affinity purification of a potential receptor. M.Sc. Thesis, National University of Ireland, Cork. Deeth, H. C. (2006). Lipoprotein lipase and lipolysis in milk. International Dairy Journal, 16, 555–562. Deeth, H. C., & Fitz-Gerald, C. H. (1995). Lipolytic enzymes and hydrolytic rancidity in milk and milk products. In P. F. Fox (Ed.), Advanced dairy chemistry (Lipids, Vol. 2, pp. 247–308). London: Chapman & Hall. Deeth, H. C., & Fitz-Gerald, C. H. (2006). Lipolytic enzymes and hydrolytic activity. In P. F. Fox & P. L. H. McSweeney (Eds.), Advanced dairy chemistry (3rd ed., Vol. 2, pp. 481–555). New York, NY: Kluwer Academic-Plenum. Dwivedi, B. K. (1973). The role of enzymes in food flavors. Part I. Dairy products. CRC Critical Reviews in Food Technology, 3, 457–478. Enroth, C., Eger, B. T., Okamoto, K., Nishino, T., Nishino, T., & Pai, E. (2000). Crystal structures of bovine xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of con- version. Proceedings of the National Academy of Sciences of the United States of America, 97, 10723–10728. Everse, J., Everse, K. E., & Grisham, M. B. (Eds.). (1991). Peroxidases in chemistry and biology (Vol. I & II). Boca Raton, FL: CRC Press. Farkye, N. Y. (2003). Indigenous enzymes in milk; other enzymes. In P. F. Fox & P. L. H. McSweeney (Eds.), Advanced dairy chemistry (Proteins 3rd ed., Vol. 1, pp. 571–603). New York, NY: Kluwer Academic-Plenum. Flynn, N. K. R. (1999). Isolation and characterization of bovine milk acid phosphatase. M.Sc. Thesis, National University of Ireland, Cork. Fox, P. F. (2003a). Significance of indigenous enzymes in milk and dairy products. In J. R. Whitaker, A. G. J. Voragen, & D. W. S. Wong (Eds.), Handbook of food enzymology (pp. 255–277). New York, NY: Marcel Dekker. Fox, P. F., & Kelly, A. L. (2006a). Indigenous enzymes in milk: Overview and historical aspects— Part 1. International Dairy Journal, 16, 500–516. Fox, P. F., & Kelly, A. L. (2006b). Indigenous enzymes in milk: Overview and historical aspects— Part 2. International Dairy Journal, 16, 517–532.

412 10  Enzymology of Milk and Milk Products Fox, P. F., & Morrissey, P. A. (1981). Indigenous enzymes of bovine milk. In G. G. Birch, N. Blakeborough, & K. J. Parker (Eds.), Enzymes and food processing (pp. 213–238). London, UK: Applied Science. Fox, P. F., Olivecrona, T., Vilaro, S., Olivecrona, G., Kelly, A. L., Shakeel-ur-Rheman, et al. (2003). Indigenous enzymes in milk. In P. F. Fox & P. L. H. McSweeney (Eds.), Advanced dairy chemistry Proteins (Vol. 1, Part A, 3rd ed., pp. 467–603). New York, NY: Kluwer Academic-Plenum. Fox, P. F., and Tarassuk N. P. (1968). Bovine milk lipase. 1. Isolation from skim milk. Journal of Dairy Science., 51, 826–833. Gallagher, D. P., Cotter, P. F., & Mulvihill, D. M. (1997). Porcine milk proteins: A review. International Dairy Journal, 7, 99–118. Garattini, E., Mendel, R., Romao, M. J., Wright, R., & Terao, M. (2003). Mammalian molybdo-­ flavoenzymes, an expanding family of proteins: Structure, genetics, regulation, function and pathophysiology. Biochemical Journal, 372, 15–32. Got, R. (1971). Les enzymes des laits. Annal Nutr l’Aliment, 25, A291–A311. Groves, M. L. (1971). Minor milk proteins and enzymes. In H. A. McKenzie (Ed.), Milk proteins, chemistry and molecular biology (Vol. 1, pp. 367–418). New York, NY: Academic. Grufferty, M. B., & Fox, P. F. (1988). Milk alkaline proteinase. Journal of Dairy Research, 55, 609–630. Hamosh, M. (1995). Enzymes in human milk. In R. G. Jensen (Ed.), Handbook of milk composi- tion (pp. 388–427). San Diego, CA: Academic. Harrison, R. (2000). Milk xanthine oxidoreductase: Hazard or benefit? Journal of Nutritional and Environmental Medicine, 12, 231–238. Harrison, R. (2002). Structure and function of xanthine oxidoreductase: Where are we now? Free Radical Biology and Medicine, 33, 774–797. Harrison, R. (2004). Physiological roles of xanthine oxidoreductase. Drug Metabolism Reviews, 36, 363–375. Harrison, R. (2006). Milk xanthine oxidase: Properties and physiological roles. International Dairy Journal, 16, 546–554. Hernell, O., & Lonnerdal, B. (1989). Enzymes in human milk. In S. A. Atkinson & B. Lonnerdal (Eds.), Proteins and non-protein nitrogen in human milk (pp. 67–75). Boca Raton, FL: CRC Press. Herrington, B. L. (1954). Lipase: A review. Journal of Dairy Science, 37, 775–789. Humbert, G., & Alais, C. (1979). Review of the progress of dairy science. The milk proteinase system. Journal of Dairy Research, 46, 559–571. Hurley, M. J., Larsen, L. B., Kelly, A. L., & McSweeney, P. L. H. (2000). The milk acid proteinase, cathepsin D: A review. International Dairy Journal, 10, 673–681. IDF. (2006). Proceedings of the first IDF symposium on indigenous enzymes in milk. International Dairy Journal, 16, 499–715. Jensen, R. G., & Pitas, R. E. (1976). Milk lipoprotein lipase: A review. Journal of Dairy Science, 59, 1203–1214. Johnson, H. A. (1974). The composition of milk. In B. H. Webb, A. H. Johnson, & J. A. Alford (Eds.), Fundamentals of dairy chemistry (2nd ed., pp. 1–57). Westport, CT: AVI Publishing Co. Kato, A. (2003). Lysozyme. In J. R. Whitaker, A. G. J. Voragen, & D. W. S. Wong (Eds.), Handbook of food enzymology (pp. 971–978). New York, NY: Marcel Dekker. Kelly, A. L., & McSweeney, P. L. H. (2003). Indigenous proteinases in milk. In P. F. Fox & P. L. H. McSweeney (Eds.), Advanced dairy chemistry (Proteins 2nd ed., Vol. 1, pp. 495–521). New York, NY: Kluwer Academic-Plenum. Kitchen, B. J. (1985). Indigenous milk enzymes. In P. F. Fox (Ed.), Developments in dairy chem- istry (Lactose and minor constituents, Vol. 3, pp. 239–279). London, UK: Elsevier Applied Science. Kitchen, B. J., Taylor, G. C., & White, I. C. (1970). Milk enzymes—Their distribution and activity. Journal of Dairy Research, 37, 279–288.

References and Suggested Reading 413 Linden, G., & Alais, C. (1976). Phosphatase alkaline du lait de vache. II. Structure sous-unitaire, nature metalloproteique et parameters cinetiques. Biochimica et Biophysica Acta, 429, 205–213. Linden, G., & Alais, C. (1978). Alkaline phosphatase in human, cow and sheep milk: Molecular and catalytic properties and metal ion action. Annales De Biologie Animale, Biochimie, Biophysique, 18, 749–758. Lonnerdal, B. (1985). Biochemistry and physiological function of human milk. American Journal of Clinical Nutrition, 42, 1299–1317. Massey, V., & Harris, C. M. (1997). Milk xanthine oxidoreductase: The first one hundred years. Biochemical Society Transactions, 25, 750–755. Moatsou, G. (2010). Indigenous enzymatic activities in caprine and ovine milks. International Journal of Dairy Technology, 63, 16–31. O’Keefe, R. B., & Kinsella, J. E. (1979). Alkaline phosphatase from bovine mammary tissue: Purification and some molecular and catalytic properties. International Journal of Biochemistry, 10, 125–134. O’Mahony, J. A., Fox, P. F., & Kelly, A. L. (2013). Indigenous enzymes in milk. In P. L. H. McSweeney & P. F. Fox (Eds.), Advanced dairy chemistry (Proteins 4th ed., Vol. 1A, pp. 337–385). New York, NY: Springer. Olivecrona, T., & Bengtsson-Olivecrona, G. (1991). Indigenous enzymes in milk, Lipases. In P. F. Fox (Ed.), Food enzymology (Vol. 1, pp. 62–78). London, UK: Elsevier Applied Science. Olivecrona, T., Vilaro, S., & Bengtsson-Olivecrona, G. (1992). Indigenous enzymes in milk, Lipases. In P. F. Fox (Ed.), Advanced dairy chemistry (Proteins, Vol. 1, pp. 292–310). London, UK: Elsevier Applied Science. Olivecrona, T., Vilaro, S., & Olivecrona, G. (2003). Indigenous enzymes in milk, Lipases. In P. F. Fox & P. L. H. McSweeney (Eds.), Advanced dairy chemistry (Proteins 3nd ed., Vol. 1, pp. 473–494). New York, NY: Kluwer Academic-Plenum. Palmquist, D. L. (2006). Milk fat: Origin of fatty acids and influence of nutritional factors thereon. In P. F. Fox & P. L. H. McSweeney (Eds.), Advanced dairy chemistry (3rd ed., Vol. 2, pp. 43–92, 555). New York, NY: Kluwer Academic-Plenum. Robert, A. M., & Robert, L. (2014). Xanthine oxido-reductase, free radicals and cardiovascular disease. A critical review. Pathology and Oncology Research, 20, 1–10. Seifu, E., Buys, E. M., & Donkin, E. F. (2005). Significance of the lactoperoxidase system in the dairy industry and its potential applications: A review. Trends in Food Science and Technology, 16, 137–154. Shahani, K. M. (1966). Milk enzymes: Their role and significance. Journal of Dairy Science, 49, 907–920. Shahani, K. M., Harper, W. J., Jensen, R. G., Parry, R. M., Jr., & Zittle, C. A. (1973). Enzymes of bovine milk: A review. Journal of Dairy Science, 56, 531–543. Shahani, K. M., Kwan, A. J., & Friend, B. A. (1980). Role and significance of enzymes in human milk. American Journal of Clinical Nutrition, 33, 1861–1868. Shakeel-ur-Rehman, Fleming, C. M., Farkye, N. Y., & Fox, P. F. (2003). Indigenous phosphatases in milk. In P. F. Fox & P. H. L. McSweeney (Eds.), Advanced dairy chemistry (Proteins, Vol. 1, pp. 523–543). New York, NY: Kluwer Academic-Plenum. Sindhu, J. S., & Arora, S. (2011). Buffalo milk. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (2nd ed., Vol. 3, pp. 503–511). Oxford, UK: Academic. Tkadlecova, M., & Hanus, J. (1973). [Enzymes in cows’ milk]. Die Nahrung, 17, 565–577. Uniacke-Lowe, T., & Fox, P. F. (2011). Equid milk. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (2nd ed., Vol. 3, pp. 518–529). Oxford, UK: Academic. Whitaker, J. R., Voragen, A. G. J., & Wong, D. W. S. (2003). Handbook of food enzymology. New York, NY: Marcel Dekker. Whitney, R. M. L. (1958). The minor proteins of milk. Journal of Dairy Science, 41, 1303–1323. Yuan, Z. Y., & Jiang, T. J. (2003). Horseradish peroxidase. In J. R. Whitaker, A. G. J. Voragen, & D. W. S. Wong (Eds.), Handbook of food enzymology (pp. 403–411). New York, NY: Marcel Dekker.

414 10  Enzymology of Milk and Milk Products Exogenous Enzymes in Dairy Technology and Analysis Brown, R. J. (1993). Dairy products. In T. Nagodawithana & J. Reed (Eds.), Enzymes in food pro- cessing (3rd ed., pp. 347–361). San Diego, CA: Academic. Dekker, P. J. T., & Daamen, C. B. G. (2011). β-D-Galactosidase. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 2, pp. 276–283). Oxford, UK: Academic. El-Soda, M., & Awad, S. (2011). Accelerated cheese ripening. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 1, pp. 795–798). Oxford, UK: Academic. Fox, P. F. (1991). Food enzymology (Vol. 1 & 2). London, UK: Elsevier Applied Science. Fox, P. F. (1993). Exogenous enzymes in dairy technology—A review. Journal of Food Biochemistry, 17, 173–199. Fox, P. F. (1998/99). Acceleration of cheese ripening. Food Biotechnology 2, 133–185. Fox, P. F. (2003b). Exogenous enzymes in dairy technology. In J. R. Whitaker, A. G. J. Voragen, & D. W. S. Wong (Eds.), Handbook of food enzymology (pp. 279–301). New York, NY: Marcel Dekker. Fox, P. F., & Grufferty, M. B. (1991). Exogenous enzymes in dairy technology. In P. F. Fox (Ed.), Food enzymology (Vol. 1 & 2, pp. 219–269). London, UK: Elsevier Applied Science. Fox, P. F., & Stepaniak, L. (1993). Enzymes in cheese technology. International Dairy Journal, 3, 509–530. Guilbault, G. G. (1970). Enzymatic methods of analysis. Oxford, UK: Pergamon Press. IDF. (1998). The use of enzymes in dairying (Bulletin, Vol. 332, pp. 8–53). Brussels: International Dairy Federation. Jaros, D., & Rohm, H. (2011). Transglutaminase. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 2, pp. 297–300). Oxford, UK: Academic. Kilara, A. (2011). Lipases. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 2, pp. 284–288). Oxford, UK: Academic. Kilara, A. (1985). Enzyme-modified lipid food ingredients. Process Biochemistry, 20(2), 35–45. McSweeney, P. L. H. (2011). Catalase, glucose oxidase, glucose isomerase, and hexose oxidase. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 2, pp. 301–303). Oxford, UK: Academic. Morris, B. A., & Clifford, M. N. (1984). Immunoassays in food analysis. London, UK: Elsevier Applied Science. Mottola, N. A. (1987). Enzymes as analytical reagents: Substrate determinations with soluble and immobilized enzyme preparations. Analyst, 112, 719–727. Nagodawithana, T., & Reed, J. (Eds.). (1993). Enzymes in food processing (3rd ed.). San Diego, CA: Academic. Nelson, J. H., Jensen, R. G., & Pitas, R. E. (1977). Pregastric esterase and other oral lipases: A review. Journal of Dairy Science, 60, 327–362. Nongonierma, A. B., & FitzGerald, R. J. (2011). Proteinases. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 2, pp. 289–296). Oxford, UK: Academic. O’Sullivan, M. M., Kelly, A. L., & Fox, P. F. (2001). Effect of transglutaminase on the heat stabil- ity of milk. Journal of Dairy Science, 85, 1–7. Whitaker, J. R. (1991). Enzymes in analytical chemistry. In P. F. Fox (Ed.), Food enzymology (Vol. 2, pp. 287–308). London, UK: Elsevier Applied Science. Wilkinson, M. G., Doolan, I. A., & Kilcawley, K. N. (1911). Enzyme-modified cheese. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 1, pp. 799–804). Oxford, UK: Academic.

Chapter 11 Biologically Active Compounds in Milk 11.1  Introduction Despite a significant amount of research in many areas, the definition of a bioactive compound remains ambiguous and unclear (see Guaadaoui et al. 2014). Bioactive compounds in foods are generally regarded as components that affect biological processes or substrates and, hence, have an impact on body function or condition and ultimately health (Schrezenmeir et al. 2000). However, this definition has been refined by two caveats: (1) To be considered bioactive, a dietary component should impart a measurable biological effect at a physiologically realistic level, and (2) The bioactivity measured must have the potential to affect health in a benefi- cial manner, thus excluding potential damaging effects from the definition such as toxicity, allergenicity and mutagenicity (Schrezenmeir et al. 2000; Möller et al. 2008) Milk is secreted by all mammals for nutrition of the young; milk secretion is one of the distinguishing characteristic of mammals (~4,500 species). Milk is required to meet the nutritional and physiological requirements of neonates which are born at different, species-specific, states of development and have different growth rates; therefore, the milk of different species differs in composition and, in effect, is species-­specific. The nutritional value of milk is due to the presence of lactose, proteins, lipids and inorganic elements (metals). Bioactive compounds in milk perform many functions other than nutritional, e.g., immune system, hor- mones and related compounds, antibacterial agents, enzymes (~60), enzyme inhibitors and cryptic peptides (various functions). Biologically active milk com- pounds in the form of immunoglobulins (Igs), antibacterial peptides, antimicrobial proteins, oligosaccharides (OSs) and lipids protect neonates and adults against © Springer International Publishing Switzerland 2015 415 P.F. Fox et al., Dairy Chemistry and Biochemistry, DOI 10.1007/978-3-319-14892-2_11

416 11  Biologically Active Compounds in Milk Lipids Lactose and Enzymes Immunoglobulins Oligosaccharides Bioactive components Lactoferrin of milk Growth factors Caseins and Vitamins and cytokinins whey proteins Peptides Fig. 11.1  The principal groups of bioactive compounds in milk (from Park 2009) pathogens and illnesses. Gobbetti et al. (2007) categorised the bioactivity of milk compounds into 4 major groups: 1. Gastrointestinal development, activity and function 2 . Infant development 3 . Immunological development and function 4. Microbial activity, including antibiotic and probiotic action A schematic representation of the major biologically active compounds in milk is shown in Fig. 11.1. In this chapter, the great diversity of biologically active com- pounds in milk will be described. While most scientific work has been carried out on the bioactive compounds in bovine milk, reference is made throughout to bioac- tive components in human milk and their significance in infant growth and development. For neonates, colostrum produced during the first 48–72 h post-partum is the only source of essential nutrients and protection against microbial infection via Igs. For several further months, breast-fed infants benefit from bacteria, such as bifido- bacteria, in the gut which reduce enteric disorders (Zinn 1997). Other compounds also provide the neonate with immunological protection and facilitate the develop- ment of neonatal immune competence; the two major categories are cytokines, which are not produced efficiently by the neonate, and peptides (Politis and Chronopoulou 2008). Individually or collectively, milk proteins, together with other bioactive compo- nents, influence the health of the neonate and may influence the health and milk production capacity of the lactating female (Zinn 1997). Women who lactate have a significantly reduced risk of breast cancer compared to those who do not (Freudenheim et al. 1994). Some milk proteins [e.g., the feedback inhibitor of lacta- tion (FIL)] may influence total milk production (Peaker and Wilde 1996). The advantages of mother’s milk may extend well beyond the first few hours post-p­ artum

11.2  Bioactive Milk Lipids 417 and may have significant consequences later in life. It has been reported that human infants who are breast-fed for 6 months have fewer health-related problems later in life than formula-fed infants, including a reduced risk of allergies (Saarinen and Kajosaari 1995), respiratory and gastrointestinal disorders (Koletzko et al. 1989), childhood lymphomas (Davies 1988; Schwartzbaum et al. 1991), type-I diabetes (Borch-Johnsen, et al. 1984) and pre- and post-menopausal cancer (Byers et al. 1985). The act of suckling may influence the growth rate of neonates (Zinn 1997). It has been reported that piglets nursed by the sow grow more slowly than those raised on milk replacer and it is postulated that the sow’s milk limits the availability of nutrients to the piglet although it is not known if the slower growth rate positively correlates with long-term health benefits (Boyd et al. 1995). 11.2  Bioactive Milk Lipids The principal function of milk lipids is as an energy source but components of the lipid fraction serve some specific biological functions; the significance of the essen- tial fatty acids, linoleic and linolenic acids, and the fat-soluble vitamins is well known. Bioactive milk lipids include, triacylglycerols (triglycerides), fatty acids, sterols and phospholipids. Anticarcinogenic activity has been attributed to conjugated linoleic acid (CLA), sphingomyelin, butanoic acid, ether lipids (plasmalogens), β-carotene, vitamin A (retinol) and vitamin D; the subject is reviewed by Parodi (1999). 11.2.1  Medium Chain Fatty Acids Medium chain fatty acids (MCFAs), containing six to ten carbons, are very different from long chain fatty acids in terms of their chemical and physical properties, e.g., MCFAs do not require binding to proteins for transport (Marten et al. 2006). In bovine milk, MCF’s make up 4–12 % of all fatty acids (Jensen 2002). MCFAs are hydrolysed rapidly and completely in the body after absorption across the epithelial barrier (Bach and Babayan 1992). Research has focussed recently on the ability of MCFA’s to reduce body weight and particularly body fat and their usefulness as supplements in functional foods is the subject of many studies (for review see Marten et al. 2006). 11.2.2  C onjugated Linoleic Acid More recently, the importance of CLA [9, 11- or 10,12-octadecadienoic acid (8 cis, trans isomers)] has been highlighted. Ruminant milk and body lipids contain a rela- tively high level of CLA (0.24–2.8 % of total fatty acids in milk fat), being pro- duced in the rumen by incomplete biohydrogenation of n-6 octadecadienoic acid

418 11  Biologically Active Compounds in Milk (LA) (see Whigham et al. 2000; Bauman and Lock 2006; Collomb et al. 2006); CLA can be increased by feeding cows with polyunsaturated fatty acid (PUFA) -rich oils (Stanton et al. 2003). CLA is also formed in mammary tissue from trans vaccenic acid (Aminot-Gilchrist and Anderson 2004). CLA has several desirable effects in the diet, some of the positive health effects attributed to it include sup- pression of carcinogenesis, anti-obesity agent, modulator of the immune system and control of atherogenesis and diabetes. Current research on CLA is focussed on its potential in the treatment or prevention of type-2 diabetes and prevention of heart disease and other health problems (see Aminot-Gilchrist and Anderson 2004). 11.2.3  P olar Milk Lipids Milk contains several biologically important polar lipids including phospholipids and sphingolipids (glycosylceramides), which are located primarily in the milk fat globule membrane (MFGM). Sphingolipids are a class of lipids with a backbone of sphingoid bases and a set of aliphatic amino alcohols, including sphingosine, which are involved in signal transmission and cell recognition. They protect cell surfaces against harmful environmental factors by forming mechanically stable and chemi- cally resistant outer leaflets of the plasma membrane ‘lipid bilayer’ (Rombaut and Dewettinck 2006). Both phospholipids and sphingolipids are regarded as being highly bioactive, with anti-cancer, bacteriostatic and cholesterol-lowering proper- ties. The nutritional and technological properties of phospholipids and sphingolip- ids were reviewed by Rombaut and Dewettinck (2006). 11.2.4  Fatty Acids with Significant Bioactivity The most abundant fatty acids in human milk are oleic (C18:1), palmitic (C16:0), lin- oleic (C18:2, ω-6) and α-linolenic (C18:3, ω-3). Some unsaturated fatty acids in human milk may provide protection against microorganisms, probably by disrupting viral envelopes, while others defend against enteric parasites, e.g., Giardia lamblia, a flagellated protozoan parasite that colonizes and reproduces in the small intestine, causing giardiasis (Thormar and Hilmarsson 2007). Both n-6 and n-3 fatty acids are essential in human metabolism as compo- nents of membrane phospholipids, precursors of eicosanoids, ligands for mem- brane receptors and transcription factors that regulate gene expression. The importance of LA (n-6 C18:2) has been known for many years but the significance of α-linolenic acid (ALA, n-3 C18:3) was not recognized until the late 1980s and has since been identified as a key component of the diet for the prevention of atopic dermatitis (Horrobin 2000). LA and ALA are not interconvertible but are the parent acids of the n-6 and n-3 series of long-chain polyunsaturated fatty acids, respectively [e.g., n-6 C20:4, arachidonic acid (AA); n-3 C20:5, eicosapen- taenoic acid (EPA) and n-3 C22:6, docosahexaenoic acid (DHA)] which are com- ponents of cellular membranes and precursors of other essential metabolites

11.2  Bioactive Milk Lipids 419 such as prostaglandins and prostacyclins (Cuthbertson 1999; Innis 2007). DHA and AA are now recognised as being crucial for normal neurological develop- ment (Carlson 2001). Both DHA and EPA are critical for the normal growth and development of the central nervous system and the retina (Uauy et al. 1990). Humans have evolved on a diet with a ratio of n-6 to n-3 fatty acids of ~ 1:1 but current Western diets have a ratio of 15:1 to 16.7:1. As a species, humans are generally deficient in n-3 fatty acids and have excessive levels of n-6 which is associated with the pathogenesis of cardiovascular, cancerous, inflammatory and autoimmune diseases (Simopoulos 2002). Butyric acid (C4:0) represents ~10 % of all fatty acids in bovine milk and is generated from carbohydrates by bacteria in the rumen, then transported via the blood to the mammary gland where it is reduced to butanoic acid (Jensen 1999). Butyrate is reported to exert many effects on intestinal function (Hamer, et al. 2008), especially colonic mucosa. Its anti-proliferate, anti-inflammatory and apoptotic properties were reviewed by Mills et al. (2009). Ingested milk butyrate does not reach the large intestine in humans but it can undergo lipase-mediated hydrolysis in the stomach which enables absorption by the proximal small intestine from where it is transported to the liver (Parodi 1997a). 11.2.5  G angliosides The name ganglioside was first applied by the German scientist Ernst Klenk in 1942 to lipids isolated from ganglion cells of the brain. A ganglioside is a molecule com- posed of a glycosphingolipid (ceramide and OS) with one or more residues (e.g., N-acetylneuraminic acid, NANA) linked to the OS chain. More than 60 ganglio- sides are known, which differ from each other mainly in the position and number of NANA residues. Gangliosides are components of the cell plasma membrane that modulates cell signal transduction events and appear to be concentrated in lipid rafts. Gangliosides are very important molecules in immunology. The OSs on gan- gliosides extend beyond the surface of cell membranes and act as distinguishing surface markers that can serve as specific determinants in cellular recognition and cell-to-cell communication. These carbohydrate head groups also act as specific receptors for certain pituitary glycoprotein hormones and certain bacterial protein toxins, such as cholera toxin. The functions of gangliosides as specific determinants suggest their important role in the growth and differentiation of tissues as well as in carcinogenesis. As well as bovine and human milk, buffalo and goat milk also con- tain gangliosides (Guo 2012; Park 2012). 11.2.6  Milk Fat Globule Membrane The MFGM contains many bioactive glycoproteins and glycolipids. Many of the indigenous enzymes in milk are concentrated in the MFGM. The glycoproteins in the MFGM of human, rhesus monkey, chimpanzee, dog, sheep, goat, cow, grey

420 11  Biologically Active Compounds in Milk seal, camel, horse and alpaca have been studied; large intra- and inter-species dif- ferences have been found (see Keenan and Mather 2006). Very highly glycosylated proteins occur in the MFGM of primates, horse, donkey, camel and dog. Long (0.5–1 μm) filamentous structures, comprised of mucins (highly glycosylated pro- teins), extend from the surface of the fat globules in equine and human milk (Welsch et al. 1988). These filaments dissociate from the surface into the milk serum on cooling and are lost on heating. For unknown reasons, the filaments on bovine milk fat globules are lost much more easily than those in equine or human milk. The filaments facilitate the adherence of fat globules to the intestinal epithe- lium and probably improve the digestion of fat (Welsch et al. 1988). The mucins prevent bacterial adhesion and may protect mammary tissue against tumours (Patton 1999). Mucin (MUC1), lactadherin and butyrophilin are the principal bio- active components of the human MFGM glycoprotein fraction. MUC1 in human milk is reported to bind rotavirus; the glycoprotein, lactadherin, also binds rotavi- rus but in a different manner (Yolken et al. 1992). Lactadherin is especially resis- tant to degradation in the neonatal stomach and is abundant in the gastrointestinal tract of (GIT) breast-fed infants. Another specific protein isolated from the MFGM called fatty-acid binding protein (FABP) has been shown to inhibit some breast cancer cell lines (Spitsberg et al. 1995). Butyrophilin, acidophilin and xanthine oxidoreductase have been identified in bovine, human and equine MFGMs. All three proteins in equine milk appear to be similar to the corresponding proteins of the human MFGM, as does lactadherin which shares 74 % identity with that of the human lactadherin (Barello et al. 2008). Both xanthine oxidoreductase and acidophilin are involved in fat globule secretion with butyrophilin while lactadherin is thought to have a protective function against rotavirus in the intestinal tract (Barello et al. 2008). The bioactivity and associated health benefits of the MFGM were reviewed by Spitsberg (2005). 11.2.7  P hospholipids The three principal phospholipids found in milk are sphingomyelin, phosphatidyl choline and phosphatidyl ethanolamine; all three are involved in many cellular processes including, growth and development and myelination of the central ner- vous system (Oshida et al. 2003 a, b). Sphingomyelin has shown some anticancer effects (Parodi 2001), inhibition of the absorption of cholesterol in the intestine of rats (Noh and Koo 2004) and activation and regulation of the immune system (Cinque et al. 2003). 11.3  B ioactive Milk Carbohydrates Bioactive carbohydrates in milk include monosaccharides (glucose and galactose), disaccharides (lactose) and oligosaccharides (OSs).

11.3  Bioactive Milk Carbohydrates 421 11.3.1  Lactose Lactose is the principal carbohydrate in the milk of most mammals but all contain other carbohydrates, e.g., galactosamine, glycoproteins and especially OSs. As well as being a major energy source for the neonate, lactose affects bone mineral- ization during the first few months post-partum as it stimulates the intestinal absorption of calcium (Schaafsma 2003). Heat treatment of lactose produces lact- ulose, an osmotic laxative and bifidus factor, which is added to infant formulae and lacto-­oligosaccharides are also produced from lactose through enzymatic processes. 11.3.2  Oligosaccharides OSs are polymers containing three to nine simple sugars which are present in all mammalian milk (Chap. 2). The OSs in milk are important protective factors and inhibit the binding of enteropathogenic E. coli, Campylobacter jejuni and Streptococcus pneumonia to target cells (Shah 2000). Fucosylated OSs, glycopro- teins and glycolipids are reported to protect the human infant against enterotoxi- genic E. coli (Newburg et al. 1990; Bode 2006). There is a very high concentration of OSs in human (>15 g L−1; >130 OSs) and in elephant and bear milk. The milk of monotremes and marsupials contains very little free lactose but mainly OSs. Colostrum of all species is especially rich in OSs (see Chap. 2). OSs are relatively resistant to hydrolysis by β-galactosidase and are indigestible in the infant GIT, i.e., they serve as soluble fibre and promote the growth of bifido- bacter. They may be absorbed from lower intestine by pinocytosis and are hydro- lysed by lysosomal enzymes and the monosaccharides catabolised for energy. OSs are a major source of energy for monotremes, marsupials and bears, i.e., species with immature (altrical) young. In human milk, the concentration of OSs, especially fucosyloligosaccharides, varies significantly over the course of lactation and between individual mothers, suggesting that the protective effects of milk against intestinal pathogens vary among individuals (Chaturvedi et al. 2001). Galactose and especially sialic acids are required for the biosynthesis of glyco- proteins and glycolipids, which are essential for brain development (see Urashima et al. 2009, 2011, 2014). In humans, the highest concentration of sialic acid (as N-acetylneuraminic acid) occurs in the brain where it forms an integral part of g­ anglioside structure in synaptogenesis and neural transmission. Human milk has an exceptionally high level of sialic acids attached to the terminals of free OSs and, while the metabolic fate and biological role are largely unknown, it has been postu- lated that it confers a developmental advantage to breast-fed infants over bottle-fed infants (Wang and Brand-Miller 2003).

422 11  Biologically Active Compounds in Milk 11.3.3  Bifidus Factors Bifidobacterium is a genus of Gram-positive microorganisms which are ubiquitous endosymbiotic inhabitants of the GIT, vagina and mouth of mammals, including humans. It has been recognized for many years that breast-fed babies are more resis- tant to gastroenteritis than bottle-fed babies. This is undoubtedly a multifactorial phenomenon, including better hygiene, more appropriate milk composition, several antibacterial systems [especially Igs, lysozyme (Lyz), lactoferrin (Lf), vitamin-­ binding proteins and lactoperoxidase (LPO)], and a lower intestinal pH. The mean pH of the faeces of breast-fed infants is ~5.1 while that of bottle-fed infants is ~6.4 due, in part, to the difference in composition between human and bovine milk—the former contains much less protein and phosphate and therefore has a lower buffering capacity but the intestinal microflora of breast-fed and bottle-f­ed differ widely; the microflora in the faeces of breast-fed infants is mainly B. bifidum, while that of bottle-fed infants is mainly B. longum, with lower numbers of B. bifidum. Bifidobacteria are acid producers and their growth in breast-fed infants is pro- moted by the bifidus factors in human milk. The growth of bifidobacteria is stimulated by several factors, but the principal one in this case is N-acetylglucosamine- containing saccharides (Bifidus Factor 1) which is present at high levels in human milk and colostrum and bovine colostrum but not in bovine, goat or sheep milk. Human milk also contains several non-dialysable bifidus-p­ romoting factors which are glycoproteins, referred to as bifidus factor 11. Many of the glycoproteins have been isolated and characterized (see Fox and Flynn 1992). 11.3.4  F ucose L-Fucose (6-deoxy-l-galactose) is a monosaccharide that is a common component of many N-and O-linked glycans and glycolipids produced by mammalian cells. Two structural features distinguish fucose from other six-carbon sugars present in mammals, the lack of a hydroxyl group on the carbon at the 6-position (C-6) and the l-configuration (Becker and Lowe 2003). Human milk OSs are highly fucosylated, whereas fucosylated OSs are either absent, or present at very low concentrations, in bovine milk (Finke et al. 2000; Tao et al. 2008; Nwosu et al. 2012). Saito et al. (1987) reported the presence of fucosylated OSs in bovine colostrum. By contrast, bovine milk is highly sialylated, whereas human milk is not (Tao et al. 2008; Nwosu et al. 2012). Free fucose has been reported not to occur in human milk (Barfoot et al. 1988) although later studies reported its presence, albeit at a low concentration, along with free N-acetylneuraminic acid and N-aceetylhexosamine (Newburg and Weiderschain 1997; Wiederschain and Newburg 2001). α-l-fucosidase activity has been reported in human milk and activity increases over the course of lactation (Newburg and Weiderschain 1997); however, even after 16 h storage at normal body temperature, free fucose levels in human milk represent only ~ 5 % of available bound fucose, although it may be significant in preventing intestinal infection in the neonate (Wiederschain and Newburg 2001).

11.4 Vitamins 423 11.4  Vitamins Vitamins are vital bioactive compounds in milk as they are essential, in minute amounts, for normal physiological functions and are not synthesized by the host in adequate amounts to meet such needs (Combs 2012). Ruminants obtain vitamins from feed and can also absorb some synthesized by intestinal microorganisms; this does not occur in humans and only a small amount of vitamin K is reabsorbed after synthesis by bacteria in the colon (Nohr 2011). Milk is an important source of vitamins A and C, thiamine, biotin (B7), riboflavin (B2), pyridoxine and cobalamin (B12). Table 11.1 shows Table 11.1  Average quantity of vitamins in bovine and human milk with recommended daily allowances (RDA) and approximate % of RDA supplied by bovine milk Vitamin Bovine Human RDAa ~ RDA (L−1) (L−1) Physiological function % in 1 L bovine milk Fat-soluble vitamins Retinol (A), mg 0.31 0.6 Visual pigments; epithelial 1 38 cell differentiation Cholecalciferol 0.2 0.3 Calcium homostasis; bone 5–10 10 (D3), μg mineralization; insulin release α-Tocopherol (E), mg 0.9 3.5 Membrane antioxidant 12 10 Phylloquinone 0.6 44 (K), mg 0.15 Blood clotting; calcium 90– metabolism 120 μg Water-soluble vitamins Ascorbic acid (C), mg 20 38 Formation of collagen and 60–75 25 carnitine Thiamine (B1), mg 0.4 0.16 Co-enzyme for 1–1.2 33 decarboxylation of 2-keto acids Riboflavin (B2), mg 1.9 0.3 Co-enzyme in redox reactions 1.2–1.4 139 of fatty acids and TCA cycle Niacin (B3), mgb 0.8 2.3 Co-enzyme for several 13–17 53 dehydrogenases Pantothenic (B5), mg 0.36 0.26 Co-enzyme in fatty acid 6 70 metabolism Pyridoxine (B6), mg 0.4 0.06 Co-enzyme in amino acid 1.2–1.5 39 metabolism Biotin (B7), μg 20 7.6 Co-enzyme for 30–60 100 carboxylations Folate (B9), mg 0.05 0.05 Co-enzyme in single carbon 400 μgc 13 metabolism Cobalamin (B12), μg 4 1.0 Co-enzyme in metabolism of 3 167 propionate, the amino acids and single-carbon units Data compiled from Schaafma (2003), Combs (2012), Morrissey and Hill (2009) and Nohr and Biesalski (2009) aValues depend on age and sex bNiacin (mg equivalents per day); 1 mg niacin equivalent = 60 mg tryptophan cCalculated for sum of folates in normal nutrition

424 11  Biologically Active Compounds in Milk comparative values for the principal vitamins of bovine and human milk and includes the percentage of the recommended daily amount present in bovine milk. In compari- son to bovine milk, human milk contains more vitamins A, E and C but less K, thia- mine, riboflavin and pyridoxine. Vitamins in milk are discussed in detail in Chap. 6. 11.5  Bioactive Milk Proteins Casein and whey proteins have been found to be increasingly important for physi- ological and biochemical functions and play a crucial role in human metabolism and health (Korhonen and Pihlanto-Leppälä 2004; Gobbetti et al. 2007). The prin- cipal function of the caseins is in nutrition as a source of amino acids, Ca2+ and Pi for the neonate; however, their amino acid sequences contain cryptic peptides which are biologically active when released by proteolysis. The major whey proteins α-lactalbumin (α-La), β-lactoglobulin (β-Lg) and immunoglobulins (Igs), have important biological roles. Many bioactive peptides, especially those from caseins, act as antioxidants in reducing cholesterol and blood pressure, others have anticar- cinogenic, anti-inflammatory, immunomodulatory, antimicrobial and wound-­ healing properties and provide protection for tooth enamel. The concentrations of casein, whey proteins and other biologically active proteins in bovine and human milk are shown in Table 11.2. 11.5.1  C aseins About 80 % of the protein of bovine milk is casein (see Chap. 4) which is primarily a source of essential amino acids and bioactive peptides, as well as a carrier of cal- cium and phosphate, for the neonate. In human milk, casein constitutes 20–30 % of total protein (Hambræus 1984). The casein fraction of most species consists of four gene products: αs1-, αs2-, β- and κ-caseins, of which the first three are calcium sensi- tive; the approximate proportions of each casein in bovine milk are 38, 11, 38 and 13 %, respectively. In other species, not all of these types of casein are present and the relative and absolute concentrations of caseins differ (Dalgleish 2011). In human milk, >85 % of the casein is β-casein (β-CN) and there is little (Rasmussen et al. 1995) or no αs-casein (α-CN, see Hambræus and Lönnerdal 2003). The amino acid sequences of the individual caseins are not well conserved between species (Martin et al. 2013). The biological function of the caseins lies in their ability to form mac- romolecular structures, casein micelles, which transfer large amounts of calcium to the neonate with a minimal risk of pathological calcification of the mammary gland. The high level of calcium in milk is important for the development, strength and density of bones in children and in the prevention of osteoporosis in adults. Calcium also reduces cholesterol absorption and controls body weight and blood pressure. A possible role of calcium in the prevention of colon cancer has been investigated and

11.5  Bioactive Milk Proteins 425 Table 11.2  The concentration of the major milk proteins of bovine and human milk and their physiological functions Protein Bovine Human Function Total protein milk (g L−1) milk (g L−1) Total casein 34 9 Ion carriers (Ca, PO4, Fe, Zn, Cu); 26 2.4 precursors of bioactive peptides αs1-Casein Precursor of bioactive peptides αs2-Casein 10.7 0.77a Precursor of bioactive peptides β-Casein 2.8 – Precursor of bioactive peptides κ-Casein 8.6 3.87 Precursor of CMP: antiviral; bifidogenic Total whey protein 3.3 0.14 β-Lactoglobulin 6.3 6.2 Retinol carrier; binding fatty acids; 3.2 – antioxidant α-Lactalbumin Synthesis of lactose; Ca carrier; 1.2 2.5 immunomodulation; anticarcinogenic Serum albumin Proteose peptone 0.4 0.48 Not characterised? Total immunoglobulins 1.2 0.8 Immune protection IgG1,2 0.8 0.96 Immune protection IgA 0.65 0.03 Immune protection IgM 0.14 0.96 Immune protection Lactoferrin 0.05 0.02 Antimicrobial; antioxidative; 0.1 1.65 immunomodulation; anticarcinogenic Lysozyme Antimicrobial; synergistic effect with 126 × 10−6 0.34 lactoferrin and immunoglobulins Lactoperoxidase Antimicrobial Miscellaneous 0.03 1.1 0.8 Adapted from Shah (2000) and Uniacke-Lowe and Fox (2012) aSee Uniacke-Lowe et al. 2010 it is hypothesized that, as bile salts are one of the main promoters of colon cancer, ingestion of milk, which provides calcium phosphate to bind bile salts, may prevent their toxic effect (van der Meer et al. 1991). 11.5.2  Whey Proteins The major whey proteins in bovine milk are β-Lg, α-La, Igs, blood serum albumin (BSA), lactoferrin (Lf) and lysozyme (Lyz), which are discussed in Chap. 4. Except for β-Lg, all these proteins are also present in human milk; however, the relative amounts of the whey proteins differ considerably between these milks. Many whey proteins have physiological properties including, metal-binding, immunomodula- tory, growth factor activity and hormonal activity. The principal anti-microbial agent in bovine milk is Lyz and to a lesser extent Lf (the latter predominates in

426 11  Biologically Active Compounds in Milk human milk (Table 11.2)). Both Lf and Lyz are present at low levels in bovine milk, in which Igs are the main defense against microbes (Malacarne et al. 2002). Together, IgA, IgG, IgM, Lf and Lyz provide the neonate with immune and non-­ immune protection against infection (Baldi et al. 2005). 11.5.2.1  Bioactivity of  β-Lactoglobulin β-Lg is the major whey protein in the milk of ruminants and is also present in milk of monogastrics and marsupials, but is absent from the milk of humans, camels, lagomorphs and rodents (see Chap. 4). Although several biological roles for β-Lg have been proposed, e.g., facilitator of retinol uptake and an inhibitor, modifier or promoter of enzyme activity, conclusive evidence for a specific biological function of β-Lg is not available (Sawyer 2003; Creamer et al. 2011). β-Lg binds retinol and that of many species, but not equine or porcine, binds fatty acids also (Pérez et al. 1993). During digestion, milk lipids are hydrolysed by pre-duodenal lipases, greatly increasing the amount of free fatty acids which could potentially bind to β-Lg, dis- placing any bound retinol, and implying that fatty acid metabolism, rather than reti- nol transport, is the more important function of β-Lg (Pérez and Calvo 1995). Bovine β-Lg is very resistant to peptic digestion and can cause allergenic reactions on consumption. Resistance to digestion may not be uniform among species; ovine β-Lg is reported to be far more digestible than bovine β-Lg (El-Zahar et al. 2005). 11.5.2.2  Bioactivity of  α-Lactalbumin α-La is a modifier of UDP-galactosyl transferase and regulates lactose biosynthesis (see Chap. 2). There is a substantial concentration in milk, ~ 4 % of total protein in bovine milk and ~ 25 % of total protein in human milk. α-La, a unique milk protein, is homologous with c-type lysozymes. It is a calcium metalloprotein, in which the Ca2+ plays a crucial role in folding and structure and has a regulatory function in the synthesis of lactose (Larson 1979; Brew 2003, 2013; Neville 2009). A high molecu- lar weight form of α-La isolated from acid-precipitated human casein causes apop- tosis of tumour cells, the native protein has no such effect but can be converted to an active anti-tumour form, HAMLET [human α–La made lethal to tumour cells] by reaction with oleic acid and conditions in an infant’s stomach can cause this change (Svensson et al. 2003). Bovine α-La can also be transformed to an anti-tumour agent, BAMLET [bovine α–La made lethal to tumour cells]. The α-La/oleic acid complex and its cytotoxic activity was reviewed by Jøhnke and Petersen (2012). 11.5.2.3  Immunoglobulins The concentration of Igs is significantly elevated in the colostrum of all ruminants (10 % of total N vs 3 % in mature milk, Fox and Kelly 2003) and equids as maternal Igs are passed from mother to neonate after birth when the small intestine is capable

11.5  Bioactive Milk Proteins 427 of absorbing intact proteins. After a few days, the gut ‘closes’ and further significant passage of proteins is prevented and within 2–3 days, the serum level of IgG in the neonate is similar to adult levels (Widdowson 1984). In contrast, in utero transfer of Igs occurs in humans and in some carnivores Igs are passed to the newborn both before and after birth. The milk of species that provide pre-natal passive immuniza- tion tends to have relatively small differences in protein content between colostrum and mature milk compared to species that depend on post-natal passage of maternal Igs. In the latter cases, of which all ungulates are typical, colostrum is rich in Igs and there are large quantitative differences in protein content between colostrum and mature milk (Langer 2009). Chapter 4 provides a detailed description of Igs. 11.5.2.4  Lactoferrin Lf is an iron-binding glycoprotein, comprised of a single polypeptide chain of MW ~78 kDa (Conneely 2001). Lf is structurally similar to transferrin (Tf), a plasma iron transport protein, but has a much higher (~300-fold) affinity for iron (Brock 1997). Lf is not unique to milk but is especially abundant in colostrum, with small amounts in tears, saliva and mucus secretions and in the secondary granules of neu- trophils. The expression of Lf in the bovine mammary gland is dependent on prolac- tin (Green and Pastewka 1978); its concentration is very high during early pregnancy and involution and is expressed predominantly in the ductal epithelium close to the teat (Molenaar et al. 1996). Human and bovine milk contain 1.65 g and 0.1 g Lf per L, respectively (Table 11.1). Shimazaki et al. (1994) purified Lf from equine milk (~0.6 g per L) and com- pared its iron-binding ability with that of human and bovine Lfs and with bovine Tf. The iron-binding capacity of equine Lf is similar to that of human Lf and higher than that of bovine Lf and Tf. Various biological functions have been attributed to Lf but its exact role in iron-binding in milk is unknown and there is no relationship between the concentrations of Lf and Tf and the concentration of iron in milk (human milk is very rich in Lf but low in iron) (Masson and Heremans 1971). Lf is a bioactive protein with nutritional and health-promoting properties (Baldi et al. 2005). Bacterial growth is inhibited by its ability to sequester iron (chelated Fe is unavailable to intestinal microorganisms) and also to permeabilize bacterial cell walls by binding to lipopolysaccharides through its N-terminus. Lf can inhibit viral infection by binding tightly to the envelope proteins of viruses and is also thought to stimulate the establishment of a beneficial microflora in the GIT (Baldi et al. 2005). Ellison and Giehl (1991) suggested that Lf and Lyz work synergisti- cally to effectively eliminate Gram-negative bacteria; Lf binds OSs in the outer bacterial membrane, thereby opening ‘pores’ for Lyz to hydrolyse glycosidic link- ages in the interior of the peptidoglycan matrix. This synergistic process leads to inactivation of both Gram-negative bacteria, e.g., E. coli (Rainhard 1986) and Gram-positive bacteria, e.g., Staph. epidermis (Leitch and Willcox 1999). A proteo- lytic digestion product of bovine and human Lf, lactoferricin, has bactericidal activ- ity (see below and Bellamy et al. 1992). Bovine and human Lf are reported to have

428 11  Biologically Active Compounds in Milk antiviral activity and act as growth factors (Lönnerdal 2003, 2013). Lf in human milk is reported to increase the production and release of cytokines such as, IL-1, IL-8, tumour necrosis factor α, nitric oxide and granulocytic-macrophage colony- stimulating factor which may have a positive effect on the immune system (Hernell and Lönnerdal 2002). Nowadays, most infant formulae are fortified with Lf (O’Regan et al. 2009; Lönnerdal and Suzuki 2013). 11.5.2.5  Serum Albumin Bovine serum albumin is the most abundant protein in the circulatory system and is multifunctional as it transports a variety of ligands, including long-chain fatty acids, steroid hormones, bilirubin and various metal ions. It is believed to enter milk via leakage through para-cellular means or via uptake with other molecules (Fox and Kelly 2003). Its physiological significance in milk is relatively insignificant as it is present at very low concentrations compared to blood plasma. 11.5.3  Vitamin-Binding Proteins Milk contains specific binding proteins for retinol (vitamin A), vitamin D, riboflavin (vitamin B2), folate and cyanocobalamin (vitamin B12). Such proteins improve the absorption of these vitamins by protecting and transferring them to receptor pro- teins in the intestine, or they may have antibacterial activity by rendering vitamins required by intestinal bacteria unavailable. The activity of these proteins is reduced or destroyed by heat treatment (see Wynn and Sheehy 2013). 11.5.3.1  R etinol-Binding Protein β-Lg binds retinol in a hydrophobic pocket and protects it against oxidation (see Chap. 4). It improves the absorption of retinol which it exchanges with a retinol-­ binding protein in the gut. It also binds fatty acids and thereby activates lipases, the significance of which is not known. Human and rodent milk lacks β–Lg; it is not known if this is significant. 11.5.3.2  Vitamin D-Binding Protein Vitamin D-binding protein (DBP), also called Gc-globulin (group-specific compo- nent), is a member of a gene family that includes serum albumin and α-fetoprotein, occurs in the plasma, ascetic fluid, cerebrospinal fluid and the surface of many cells types in most vertebrates. DBP is a 51–58 kDa multifunctional serum glycoprotein synthesized in large quantities by hepatic parenchymal cells and is secreted into the circulatory system as a monomeric peptide of 458 residues. Two binding regions are

11.5  Bioactive Milk Proteins 429 well characterized—a vitamin D/fatty acid binding domain located between resi- dues 35 and 49, and an actin binding domain between residues 350 and 403 (Malik et al. 2013). DBP circulates in amounts far in excess of normal vitamin D metabolite concentrations in blood (Haddad 1995). DBP binds vitamin D and its plasma metabolites and transports them to target tissues, it prevents polymerization of actin (G-actin) by binding its monomers and it may have significant anti-inflammatory and immunoregulatory functions (Malik et al. 2013). DBP has been detected at a low level in the milk of several species and occurs at a higher concentration in colostrum than in milk. DBP variants have attracted attention in recent years as genetic factors with major roles in several chronic disease outcomes, e.g., pancre- atic, prostate and bladder cancers (for reviews see Chun 2012 and Malik et al. 2013). 11.5.3.3  R iboflavin-Binding Protein Riboflavin-binding protein (RfBP) has been partially purified from bovine milk; it has a MW of ~38 kDa (Kanno et al. 1991). The RfBP—riboflavin complex has good antioxidant properties, similar to riboflavin bound to egg white RfBP (Toyosaki and Mineshita 1988). The RfBP in milk is probably derived from blood serum. 11.5.3.4  F olate-Binding Protein The folate-binding properties of milk have been known since the late 1960s and the involvement of a specific protein was confirmed by Ghitis et al. (1969). Later, a minor whey protein in milk was identified as having specific folate-binding proper- ties (Salter et al. 1981). A folate-binding protein (FBP) isolated from bovine and human milk is a glycoprotein with a MW of ~35 kDa (Salter et al. 1981). Its con- centration is higher (~5×) in colostrum than in milk (Nygren-Babol et al. 2004). FBP is crucial for the assimilation, distribution and retention of folic acid (Davis and Nichol 1988) and is antibacterial through reducing the availability of folate to microorganisms. The effectiveness of FBP is reduced by heat treatment of milk (Gregory 1982; Achanta et al. 2007). FBP is found in both soluble and particulate forms in human milk; ~ 22 % of the soluble form is glycosylated, providing protec- tion against digestive enzymes. FBP in human milk is thought to slow the release and uptake of folate in the neonatal small intestine to allow gradual release and absorption of folate which may improve tissue utilization (Pickering et al. 2004). FBP and its role in folate nutrition has been reviewed by Parodi (1997b), while its biochemistry and physiology were reviewed by Nygren-Babol and Jägerstad (2012). 11.5.3.5  H aptocorrin Haptocorrin (formerly called vitamin B12-binding protein) is involved in the protec- tion of acid-sensitive vitamin B12 as it passes through the GIT and is produced by the salivary glands in response to the ingestion of food. In all, three proteins are

430 11  Biologically Active Compounds in Milk involved in the uptake of vitamin B12 (cobalamin). Gastric intrinsic factor (GIF) binds free B12 released from foods on digestion and transports it to the intestine where it is transferred to another protein, transcobalamin (TC). The B12-TC com- plex and free TC are released into portal blood. Haptocorrin binds vitamin B12 to form a halohaptocorrin complex which can attach to human intestinal brush border membranes where the associated vitamin is absorbed by intestinal cells (Adkins and Lönnerdal 2001). Thus, absorption of vitamin B12 is improved by haptocorrin in the neonate and is antibacterial as protein-bound B12 is unavailable to gut microflora. Binding and inhibitory effects are reduced by heating. 11.5.4  Hormone-Binding Proteins 11.5.4.1  C orticosteroid-Binding Protein Human milk and colostrum contain two corticosteroid-binding proteins (Rosner et al. 1976); similar proteins occur in blood. The function and significance of these proteins in milk are unknown. 11.5.4.2  T hyroxine-Binding Protein The whey fraction of human milk contains a thyroxine-binding protein analogous to serum thyroxine-binding globulin at a concentration of ~0.3 mg mL−1 (Oberkotter and Farber 1984). The function of this protein in milk is unknown. 11.5.5  M etal-Binding Proteins Milk contains many metal-binding proteins (or peptides therefrom), some of which have a nutritional function while others are enzymes, for the activity of which, the metal is essential. The most important inorganic elements in milk from a bone-­ health point of view are calcium, phosphorus, magnesium, sodium, potassium and zinc (for review see Cashman 2006). The significance of milk metals on the risk factors for heart disease, diabetes, stroke and other illnesses was reviewed exten- sively by Scholtz-Ahrens and Schrezenmeir (2006). Table 11.3 shows some of the principal metal-binding peptides in milk. While casein phosphopeptides (CPPs, see 11.7.2.4) are the main carriers of metals in milk, some metal binding peptides are found in whey protein hydrolysates, α-La, β-Lg and Lf. These are not phosphory- lated peptides but metals bind to them via other sites which may be influenced by the protein conformation. α-La- and β-Lg-derived peptides have a greater affinity for iron than their parent protein (Vegarud et al. 2000).

11.6  Minor Biologically-Active Proteins in Milk 431 Table 11.3  Metal-binding peptides derived from milk proteins Protein Enzyme Phosphoresidues Net charge Metals bound Fe, Mn, Cu, Casein-derived Trypsin 2 −7 Se, Ca, Zn phosphopeptides Trypsin 7 Ca, Fe Trypsin 4 −9 αs1-CN, f 43–58 Trypsin 5 −11 Ca αs1-CN, f 43–79 Trypsin 4 Ca, Fe αs1-CN, f 59–64 Trypsin 4 −9/−8 αs1-CN, f 59−79 Trypsin 4 −6 Ca αs2-CN, f 1–21/–32 Trypsin 3 Ca, Fe αs2-CN, f 46–70 Trypsin 4 αs2-CN, f55–64 1 Cu, Ca, Zn, Fe αs2-CN, f 66–74 Pepsin, trypsin/ β-CN, f 1–25/28 chymotrypsin Fe β-CN, f 33–48 Thermolysin Fe Whey protein- Pepsin, trypsin/ derived peptides chymotrypsin Fe Pepsin/trypsin Fe α-La Trypsin Fe Trypsin β-Lg β-Lg Lf (30 kDa) Lf (40 kDa) Lf (50 kDa) CN casein, La lactalbumin, Lg lactoglobulin, Lf lactoferrin 11.6  M inor Biologically-Active Proteins in Milk Together with growth factors, minor milk proteins elicit significant effects on the growth and development of the neonatal calf as well as on maternal physiology (Wynn and Sheehy 2013). Milk contains about 100 proteins at trace levels (Table  11.4). Many of these have a biological activity. Angiogenins play several roles, especially in the vascular and immune systems. β2-Microglobulin, osteopon- tin, proteose peptone 3, lactoperoxidase (LPO), lysozyme (Lyz) and transforming growth factors (TGFs β1 and β2) all have significant biological roles in the immune system while insulin-like growth factors (IGFs 1 and 2), epidermal growth factors (EGFs) and TGF α, play important roles in facilitating maturation of the gastroin- testinal epithelium. Several minor proteins bind vitamins while others play roles in mammary gland and maternal physiological regulatory functions (e.g., leptin, feed- back inhibitor of lactation, parathyroid hormone-related peptide and relaxin). Minor proteins, including growth factors have been reviewed by many authors (see Wynn and Sheehy 2013).

432 11  Biologically Active Compounds in Milk Table 11.4  Minor biologically-active proteins in milk Protein Molecular mass (Da) Concentration in mature Source 11,636 bovine milk (mg L−1) Monocytes β2-Microglobulin 60,000 9.5 Mammary Osteopontin 28,000 3–10 Mammary 30,000 300 Proteose peptone 3 6–10 Blood 52,000 Folate-binding 16 Mammary Protein (FBP) 43,000 0.1–0.2 Blood Vitamin D-binding 14,577 Blood Protein 14,522 4–8 Blood 68,000/17,000 Mammary Vitamin B12-binding 77,000 <20 Mammary Protein 40,000 Blood, Mammary 132,000 6.0 Angiogenin-1 66,000 trace Various Angiogenin-2 Kininogen Serotransferrin α1-Acid glycoprotein Ceruloplasmin Prosaposin Enzymes (~ 60) Modified from Fox and Kelly (2003) 11.6.1  H eparin Affinity Regulatory Peptide Heparin Affinity Regulatory Peptide (HARP) is a 136-amino acid growth factor with an MW of ~18 kDa which has a high affinity for the anticoagulant glycosami- noglycan heparin and is secreted in human milk and colostrum with a threefold higher concentration in colostrum (Wynn and Sheehy 2013). Several physiological functions have been ascribed to HARP including stimulation of cell replication and chemotaxis and promoting angiogenesis both in vivo and in vitro (Papadimitriou et al. 2001). 11.6.2  C olostrinin Colostrinin is a complex of proline-rich phosphopeptides first isolated from the IgG2 fraction of ovine colostrum and containing mainly β-CN f121–138; it is also present in the colostrum of other species. It has beneficial effects on Alzheimer’s disease but it is not known how it is produced (see Bilikieweiz and Gaus 2004; Kurzel et al. 2004).

11.6  Minor Biologically-Active Proteins in Milk 433 11.6.3  β2-Microglobulin β2-Microglobulin (β2-MG) is homologous to the “constant domain” of Ig and histo- compatibility antigen (Groves and Greenberg 1982). It is probably produced in milk by intra-mammary proteolysis of somatic cells. Its MW is 11,636 Da and it contains 98 amino acid residues. It was isolated initially as a tetramer called lactollin. β2-MG occurs free in body fluids where it may help T-lymphocytes with antigen recogni- tion but its significance in milk is not known (Fox and Kelly 2003). 11.6.4  Osteopontin Osteopontin (OPN) is a highly phosphorylated glycoprotein (MW, 29,283 Da; 261 amino acid residues of which 27 are phosphoserines and 1 is phosphothreonine) with 50 Ca-binding sites (Fox and Kelly 2003). It is present in bone and many other tissues and fluids, including milk. It has many functions, including mineralization and resorption of bone and biological signalling. The significance of OPN in milk is unknown but it may be important for calcium binding or anti-infectious activity (Fox and Kelly 2003). 11.6.5  Proteose Peptone-3 Proteose peptone-3 (PP3) is a heat-stable, acid-soluble glycophosphoprotein. Unlike the other proteose peptones, PP3 is an indigenous protein in milk, occurring mainly in whey. It has a MW of 28 kDa but two proteolytic fragments (18 and 11 kDa) also occur in milk. PP3 forms an amphiphilic helix and behaves hydrophobically. It pre- vents contact between lipoprotein lipase and its substrate and thereby prevents spon- taneous lipolysis. It has been proposed that PP3 should be called lactophorin or lactoglycoporin (Girardet and Linden 1996). The biological function of PP3 in milk is unknown but it may stimulate the growth of bifidobacteria or have some involve- ment in calcium ion-binding via its phosphorylated N-terminus (Fox and Kelly 2003). 11.6.6  A ngiogenins Angiogenins induce the growth of blood vessels (angiogenesis). They have sequence homology with RNase and have RNase activity which is important for angiogene- sis. Two angiogenins (1 and 2) occur in bovine milk and blood serum; their MW is ~15 kDa. Both have strong ability to promote the growth of new blood vessels in a

434 11  Biologically Active Compounds in Milk chicken membrane assay (Fox and Kelly 2003). Their function in milk is unknown but it has been suggested that they may have a protective effect in the mammary gland or the neonatal intestine (Strydom 1998). 11.6.7  K ininogens There are two forms of kininogen in bovine milk, a high MW (>68 kDa, 626 amino acid residues, produced in the liver) and low a MW (16–17 kDa, produced in vari- ous tissues). Bradykinin, a biologically active peptide, is released from high MW kininogen by the action of the enzyme, killikrenin: Kallikrenin High MW kininogen --------® bradykinin (a 9 AA peptide) + kellidin Bradykinin is secreted into milk from the mammary gland (Fox and Kelly 2003). It is believed that kininogens in milk are different from those in blood plasma. Plasma kininogen is an inhibitor of thiol proteinases and has a role in the initia- tion of blood coagulation. Bradykinin has several functions: it affects smooth mus- cle contraction and causes vasodilation and hypotension. The function(s) of kininogen and its derivatives in milk are unknown. 11.6.8  Glycoproteins Milk contains several minor glycoproteins, one of which is M-1 glycoprotein (MW ~10 kDa). Its sugars are galactose, galactosamine and NANA and it stimulates bifidobacteria, probably via its amino sugars (Fox and Kelly 2003). Another glycoprotein, orosamucoid (α1-acid glycoprotein), has been detected in bovine colostrum but not in mature milk. It is a member of the lipocalin family with a MW of 40 kDa. It can modulate the immune system and its concentration in milk increases during inflammatory diseases, malignancy and pregnancy (Fox and Kelly 2003). 11.6.8.1  P rosaposin Prosaposin (or PSAP) is a highly conserved glycoprotein of ~66 kDa and a precur- sor for four cleavage products, saposins A, B, C and D (each contains ~80 amino acids and is glycosylated), which are required for the hydrolysis of some sphingo- lipids by specific lysosomal hydrolases. Prosapsin occurs in milk but the saposins do not. Prosaposin has been isolated from human milk (Kondoh et al. 1991; Hiraiwa